Monocyte proliferation induced by modified serum is associated with endogenous M-CSF production: evidence for involvement of a signalling pathway via scavenger receptors

It was found that human serum stored for 2 months at 4 degrees C (modified serum) induced monocyte proliferation and simultaneous macrophage colony stimulating factor (M-CSF) production by these cells in vitro. Cell number, estimated by DNA content, doubled after 10 days in culture in the presence of modified serum, while it decreased in culture with freshly thawed control serum. As the addition of more than 2.5 ng/ml of recombinant M-CSF significantly supported monocyte survival/proliferation, cells were cultured for 10 days in medium supplemented with control serum, and endogenous M-CSF production was investigated by enzyme-linked immunosorbent assay. M-CSF concentration in the supernatants was 15-30 ng/ml after 10 day in culture with modified serum, a level that might be sufficient for monocyte proliferation. The modified serum induced M-CSF from freshly isolated monocytes, while M-CSF was hardly detected in cultures supplemented with control serum. Assay for peroxidized lipid and agarose gel electrophoresis demonstrated that the modified serum contained more oxidized low density lipoproteins (LDL) than the control serum. Ligands of scavenger receptors, which are receptors for oxidized LDL, such as dextran sulphate, polyinosinic acid, heparin and acetylated LDL also significantly induced M-CSF production from human monocytes, although this was at levels below 2 ng/ml. These results indicate that serum modified by oxidation stimulates monocytes to produce M-CSF resulting in their proliferation, and that signalling via scavenger receptors is one of the mechanisms responsible for this induction of M-CSF.     

Effects of recombinant human stem cell factor (SCF) on the growth of human progenitor cells in vitro.

We have studied the effect of recombinant human Stem Cell Factor (SCF) on the growth of human peripheral blood, bone marrow, and cord blood progenitor cells in semisolid medium. While SCF alone had little colony-stimulating activity under fetal bovine serum (FBS)-deprived culture conditions, SCF synergized with erythropoietin (Epo), granulocyte/macrophage colony-stimulating factor (GM-CSF), and interleukin 3 (IL-3) to stimulate colony growth. Colony morphology was determined by the late-acting growth factor added along with SCF. Of all the combinations of growth factors, SCF plus IL-3 and Epo resulted in the largest number of mixed-cell colonies--a larger number than observed with IL-3 and Epo alone even in FBS-supplemented cultures. These results suggest that SCF is a growth factor that more specifically targets early progenitor cells (mixed-cell colony-forming cells) and has the capacity to synergize with a wide variety of other hematopoietic growth factors to cause the proliferation and differentiation of committed progenitor cells. Our studies indicate that SCF may be the earliest acting growth factor described to date.     

Interaction of serum and colony-stimulating factor for survival of a factor-dependent hemopoietic progenitor cell line

The growth in vitro of the murine myeloid cell line FDC-P1 depends on the presence of serum and a murine hemopoietic growth factor (either granulocyte/macrophage colony-stimulating factor (GM-CSF) or multipotential colony-stimulating factor (multi-CSF, IL3]. To determine the differential roles of serum and colony-stimulating factor (CSF) during the growth of FDC-P1 cultures, we investigated the kinetics of proliferation and death after withdrawal of serum or CSF, using flow cytometry to quantitate the numbers of vital and dead cells. After withdrawal of CSF, the cells died without entering a quiescent state. The life span of cultures lacking CSF increased with increasing concentrations of serum (greater than 50 h at 30% serum), and the cells kept dividing until they died. During the period of population death caused by the absence of CSF, the re-addition of CSF immediately prevented further cells from dying. After the withdrawal of serum in the presence of CSF, the cells continued to live and proliferate for weeks, but required high cell densities (much greater than 10(5)/ml), which suggests that the cells produced an active substance that can substitute for serum. Serum as well as serum-free conditioned medium from dense cultures made the survival and growth of FDC-P1 cultures independent of cell density. Without sufficient quantities of this activity, all cells of the population died within an interval that was much shorter than one cell cycle, which indicates that the factor acts throughout most of the cell cycle. The results suggest that both the CSF and the serum factor act together to permit cell survival, rather than to drive proliferation.     

Proliferative effects of purified granulocyte colony-stimulating factor (G-CSF) on normal mouse hemopoietic cells

When granulocyte colony-stimulating factor (G-CSF), purified to homogeneity from mouse lung-conditioned medium, was added to agar cultures of mouse bone marrcw cells, it stimulated the formation of small numbers of granulocytic colonies. At high concentrations of G-CSF, a small proportion of macrophage and granulocyte-macrophage colonies also developed. G-CSF stimulated colony formation by highly enriched progenitor cell populations obtained by fractionation of mouse fetal liver cells using a fluorescence-activated cell sorter, indicating that G-CSF probably acts directly on target progenitor cells. Granulocytic colonies stimulated by G-CSF were small and uniform in size, and at 7 days of culture were composed of highly differentiated cells. Studies using clonal transfer and the delayed addition of other regulators showed that G-CSF could directly stimulate the initial proliferation of a large proportion of the granulocvte-macrophage progenitors in adult marrow and also the survival and/or proliferation of some multipotential, erythroid, and eosinophil progenitors in fetal liver. However, G-CSF was unable to sustain continued proliferation of these cells to result in colony formation. When G-CSF was mixed with purified granulocyte-macrophage colony-stimulating factor (GM-CSF) or macrophage colony-stimulating factor (M-CSF), the combination stimulated the formation by adult marrow cells of more granulocyte-macrophage colonies than either stimulus alone and an overall size increase in all colonies. G-CSF behaves as a predominantly granulopoietic stimulating factor but has some capacity to stimulate the initial proliferation of the same wide range of progenitor cells as that stimulated by GM-CSF.     

Reengineering granulocyte colony-stimulating factor for enhanced stability

Granulocyte colony-stimulating factor is a long-chain cytokine that has both biological and therapeutic applications. It is involved in the production and maturation of neutrophilic progenitor cells and neutrophils and is administered to stimulate the production of white blood cells to reduce the risk of serious infection in immunocompromised patients. We have reengineered granulocyte colony-stimulating factor to improve the thermodynamic stability of the protein, focusing on enhancing the alpha-helical propensity of residues in the antiparallel 4-helix bundle of the protein. These redesigns resulted in proteins with substantially enhanced stability while retaining wild-type levels of biological activity, measured as the ability of the reengineered proteins to stimulate the proliferation of murine myeloid cells transfected with the granulocyte colony-stimulating factor receptor.     

Serum factors in chronic myeloid leukemia

Mononuclear cells from the peripheral blood of healthy test persons were cultivated in a methylcellulose medium with serum samples taken from 13 patients with chronic myeloid leukemia (CML) and with osteomyelosclerosis (OMS) as well as with serum samples of 6 healthy test persons. From evaluating the proliferation of granulopoietic cells quantitatively, conclusions were made concerning the concentrations of granulopoietic stimulating substances in these sera. In all cultures with the serum of patients the number of granulopoietic cell colonies was greater than that in cultures with the serum of normal persons. The stronger proliferation of granulopoietic precursor cells in cultures with serum of patients is seen to be due to an enhanced production of the granulocyte-macrophage colony stimulating factor (GM-CSF) by leukemic cells. The differential hemograms and curves indicating the course of leukocytes in patients are compared with the corresponding results of cultures. In patients with CML an increased output of GM-CSF will apparently influence the increase in size of the granulopoietic stem cell pool, which is evident in the steep increase of those curves indicating the course of leukocytes. In patients with OMS, however, there is a discrepancy between granulopoietic serum activity and proliferation in vivo. From these investigations the hypothesis is derived that an increased synthesis of GM-CSF in patients with CML may be one of the causes underlying hyperplastic granulopoiesis. A direct advantage of leukemic cells in proliferation cannot be derived from it.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cultured mouse marrow cell lines: Interactions between fibroblastoid cells and monocytes

Continuous cell lines were derived from primary cultures of adherent bone marrow cells from SJL/J, BALB/c, C3H/eb, RF, and nude-ICR mice. All these lines readily assumed a pure fibroblastoid appearance with the exception of the BALB/c line (MBA-14), which retained both fibroblastoid and monocytoid cells. This particular line could promote the proliferation of myeloid progenitors (CFU-C) in short-term bone-marrow cultures. The two cell types that composed the MBA-14 cell line were successfully isolated and grown separately; the monocytes as the 14M and 14M1 cell lines and the fibroblastoid cells as the 14F clones. The latter were found to be preadipocytes and accumulated fat in the absence of added hydrocortisone, in medium supplemented with fetal calf serum. Growth of the monocyte lines (14M and 14M1) was dependent upon the mononuclear phagocyte stimulator CSF-1. In the parent MBA-14 cell line the growth of monocytes seemed to depend upon stimulating factor(s) produced by the fibroblastoid cells. The 14M1 monocytes were able to process and degrade antigen as efficiently as primary macrophages. Furthermore, processed antigen produced by 14M1 cells evoked proliferative response by antigen-primed lymph-node cells. In addition to these immunological functions the 14M1 cells were capable of modulating the colony-stimulating activity and degree of adipogenesis exhibited by the fibroblastoid cells. These interactions between monocytes and fibroblastoid cells may constitute part of the mechanism controlling the activity of the hematopoietic microenvironment.     

Immature megakaryocytes in the mouse: In vitro relationship to megakaryocyte progenitor cells and mature megakaryocytes

An assay describing conditions for the maturation of single immature megakaryocytes in vitro is reported. Enriched populations of small, relatively immature megakaryocytes have been found to develop into single, mature megakaryocytes by 60 hours in semisolid agar cultures. Continued incubation of these cells did not lead to the formation of colonies within 5–7 days. Maturation was indicated by increasing cell size and cytoplasmic and acetylcholinesterase content. Factors stimulating the development of immature megakaryocytes were found in preparations of human embryonic kidney cell-conditioned media (a source of in vivo Thrombopoietic Stimulatory Factor), peritoneal exudate cell-conditioned medium, lung-conditioned medium, or bone marrow cellular sources of activity (adherent cells or cells that sediment at 5–6 mm hr^-1). Immature megakaryocytes cultured serum free responded to sources of an auxiliary megakaryocyte potentiating activity by developing into single, large megakaryocytes but did not respond to a megakaryocyte colony-stimulating factor devoid of detectable potentiator activity present in WEHl-3-conditioned medium. In contrast, serum-free proliferation of the megakaryocyte progenitor cell required both megakaryocyte colony-stimulating factor and the auxiliary potentiator activity. In the presence of megakaryocyte colony-stimulating factor alone, progenitor cells did not form colonies of easily detectable megakaryocytes. However, groups of cells comprised entirely of small acetylcholinesterase containing immature megakaryocytes were observed, thus establishing that megakaryocyte colony development passes through a stage of immature cells prior to detectable megakaryocyte development and that some acetylcholinesterase-containing cells can undergo cellular division.     

Cyclophosphamide stimulates lung fibroblasts to release neutrophil and monocyte chemoattractants

Cyclophosphamide is an alkylating antineoplastic agent used in several conditions. However, little is known about the mechanism of its pulmonary toxicity. In the present study, we determined that human lung fibroblasts release activity for neutrophils and monocytes in response to cyclophosphamide in a dose- and time-dependent manner. Checkerboard analysis revealed that both neutrophil and monocyte activities were chemotactic. The release of chemotactic activity was inhibited by lipoxygenase inhibitors and cycloheximide. Molecular-sieve column chromatography revealed that both neutrophil (NCA) and monocyte (MCA) chemotactic activities had multiple peaks. NCA was inhibited by a leukotriene B(4) receptor antagonist and anti-interleukin-8 and anti-granulocyte colony-stimulating factor antibodies. MCA was attenuated by a leukotriene B(4) receptor antagonist and anti-monocyte chemoattractant protein-1 and anti-granulocyte-macrophage colony-stimulating factor antibodies. The concentrations of interleukin-8, granulocyte colony-stimulating factor, monocyte chemoattractant protein-1, and granulocyte-macrophage colony-stimulating factor significantly increased in response to cyclophosphamide. These data suggest that lung fibroblasts may modulate inflammatory cell recruitment into the lung by releasing NCA and MCA in response to cyclophosphamide.     

Cellular responsiveness to stimulation in vitro: increased responsiveness to colony stimulating factor of bone marrow colony-forming cells treated with surface-active agents and cyclic 3'5' AMP.

Addition of low concentrations (10 ng/ml) of saponin or Tween 80 to stimulated cultures of normal mouse bone marrow in agar increased the number of granulocyte-macrophage colonies which developed. Addition of cyclic AMP or dibutyryl cyclic AMP in low concentration (10(-8) to 10(-10) M) also enhanced colony numbers although concentrations above 10(-5) M were inhibitory. enhancement was found when marrow cells were pre-treated with these agents and cultured in their absence. The agents did not stimulate colony development in the absence of colony-stimulating factor and enhancement of colony number occurred only in cultures containing a concentration of colony-stimulating factor which was sub-optimal in terms of maximum colony development. There was no indication of increased colony-stimulating factor production by treated marrow cells under the experimental conditions used to show colony enhancement. It was concluded that the agents caused an increased responsiveness of colony-forming cells to colony-stimulating factor.     

Heterogeneity of in vitro colony- and cluster-forming cells in the mouse marrow: segregation by velocity sedimentation.

C57BL bone marrow cells were separated on the basis of their sedimentation velocity at unit gravity and cell fractions cultured in agar using three types of colony stimulating factor (CSF). Colony-forming cells separated as a single peak (s equal 4.4 mm/hr) in cultures stimulated by mouse lung conditioned medium (CSFMLCM) or endotoxin serum (CSFES). Cluster-forming cells were separable into two peaks and the majority were larger than colony-forming cells (s equal 5.7 mm/hr). Partial segregation of colony-forming cells was observed according to the morphological types of colonies generated, large cells tending to generate macrophage colonies and small cells, granulocytic colonies. Large colony-forming cells were more responsive to stimulation by CSF than small cells. Human urine (CSFHU) appeared unable to proliferation of most small colony-forming cells. Colony-forming cells appear to be a highly heterogeneous population with intrinsic differences in responsiveness to CSF and with differing capacities to generate colonies whose cells differentiate to granulocytes of macrophages.     

Cytokine-mediated protein kinase C activation is a signal for lineage determination in bipotential granulocyte macrophage colony-forming cells

Granulocyte macrophage colony-forming cells (GM-CFC) have the potential to develop into either macrophages and/or neutrophils. With a highly enriched population of these cells we have found that although GM-CFC are equally responsive to macrophage colony stimulating factor (M-CSF) and stem cell factor (SCF) in terms of DNA synthesis, M-CSF stimulated the development of colonies containing macrophages in soft gel assays, while SCF promoted neutrophilic colony formation. When SCF and M-CSF were combined, mainly macrophage development was stimulated both in soft agar colony-forming assays and liquid cultures. An analysis of some potential signaling mechanisms associated with cytokine-mediated developmental decisions in GM-CFC revealed that M-CSF, but not SCF, was able to chronically stimulate phosphatidylcholine breakdown and diacylglycerol production, indicating that protein kinase C (PKC) may be involved in the action of M-CSF. Furthermore, M-CSF, but not SCF, can increase the levels of PKC alpha (PKC alpha) expression and stimulate the translocation of PKC alpha to the nucleus. When the PKC inhibitor, calphostin C, was added to GM-CFC cultured in M-CSF then predominantly neutrophils were produced, conversely PKC activators added with SCF stimulated macrophage development. The data indicate a role for PKC in M-CSF-stimulated macrophage development from GM-CFC.     

Action of inhibitor released from neutrophils and leukemic blast cells.

The concept that polymorphonuclear leukocytes, or neutrophils, play a role in feedback control of granulopoiesis has been supported by the finding in bone marrow culture studies that mature neutrophils inhibited formation of granulocytic colonies. The study described in this paper was done to investigate the mechanisms involved. With the use of a modified assay it was found that mature neutrophils released factors that reduced the proliferation of colony-forming cells in cultures stimulated by cell-free colony-stimulating factor. In myeloproliferative and myelodysplastic disorders the amount of inhibitor released by the neutrophils varied greatly. Leukemic blast cells also released inhibitor, and in some cases the amount released per cell was greater than the amount released from normal mature neutrophils. The inhibitory factors released from the neutrophils differed from those previously described in the literature in terms of mode of action and apparent molecular size.     

Nicotinic Acetylcholine Receptors Modulate Bone Marrow-Derived Pro-Inflammatory Monocyte Production and Survival

It is increasingly clear that nicotinic acetylcholine receptors (nAChRs) are involved in immune regulation, and that their activation can protect against inflammatory diseases. Previous data have shown that nicotine diminishes the numbers of peripheral monocytes and macrophages, especially those of the pro-inflammatory phenotype. The goal of the present study was to determine if nicotine modulates the production of bone marrow -derived monocytes/macrophages. In this study, we first found that murine bone marrow cells express multiple nAChR subunits, and that the α7 and α9 nAChRs most predominant subtypes found in immune cells and their precursors. Using primary cultures of murine bone marrow cells, we then determined the effect of nicotine on monocyte colony-stimulating factor and interferon gamma (IFNγ)-induced monocyte production. We found that nicotine lowered the overall number of monocytes, and more specifically, inhibited the IFNγ-induced increase in pro-inflammatory monocytes by reducing cell proliferation and viability. These data suggested that nicotine diminishes the ratio of pro-inflammatory versus anti-inflammatory monocyte produced in the bone marrow. We thus confirmed this hypothesis by measuring cytokine expression, where we found that nicotine inhibited the production of the pro-inflammatory cytokines TNFα, IL-1β and IL-12, while stimulating the secretion of IL-10, an anti-inflammatory cytokine. Finally, nicotine also reduced the number of pro-inflammatory monocytes in the bone marrow of LPS-challenged mice. Overall, our data demonstrate that both α7 and α9 nAChRs are involved in the regulation of pro-inflammatory M1 monocyte numbers.     

LOCAL AND SYSTEMIC CONTROL OF GRANULOCYTIC AND MACROPHAGE PROGENITOR CELL REGENERATION AFTER IRRADIATION

Agar cultures of C₅₇BL bone marrow cells were used to determine colony stimulating factor (CSF) and serum CSF-inhibitor levels in C₅₇BL and BALB/c mice following irradiation. Whole-body irradiation caused an acute, dose-dependent, rise in serum CSF levels and fall in CSF-inhibitor levels. The regeneration of granulocytic and macrophage progenitor cells ( in vitro CFCs) in the femur after 250 rads whole-body irradiation was preceded or paralleled by a fall in serum CSF-inhibitors and a dramatic rise in the capacity of bone-adherent cells in the marrow ('stromal cells') to produce material with colony-stimulating activity. No comparable changes were observed in the activity of marrow haemopoietic cells during regeneration or in the lungs or spleen. A similar rise in the activity of bone-adherent cells was observed in shielded femurs during regeneration of in vitro CFCs.
Regeneration of granulocytic and macrophage progenitor cells following irradiation may be regulated by fluctuations in circulating CSF-inhibitor levels and local production of CSF within the marrow cavity.     

Characterization of the human erythroid progenitor cells responsive to the erythropoietic-enhancing activity found in normal human serum

Normal human serum significantly increased the growth of erythroid colonies from bone marrow colony-forming units-erythroid (CFU-e) which were enriched by using a set of monoclonal antibodies in a panning technique. This activity was still observed in cultures of enriched CFU-e plated near the limiting cell dilution. When the addition of erythropoietin was delayed so that only early CFU-e could survive, we observed that the growth of erythroid colonies was less affected in cultures containing erythropoietin and normal serum than in those containing erythropoietin only. We have concluded from this study that normal human serum acts on in vitro erythropoiesis by directly stimulating the growth of a population of early CFU-e.     

The study of mechanisms of radioprotective action of indometofen n hematopoietic stem cells in mouse long-term bone marrow cultures

The influence of indometophen (an analog of tamoxiphen) on the dynamic content and the proliferative activity of CFUs (colony-forming units) and CFU-GM (granulocyto-macrophages precursors) and the level of colony-stimulating factor (GM-CSF) in mouse long-term bone marrow cultures were studied for 4 weeks after administration. Five days after indometophen injection the long-term cultures were exposed to irradiation with a dose of 2 Gy and on the time course of postirradiation recovery haemopoietic precursors cells and dynamic release of GM-CSF in the culture supernatants were examined. The data of this report suggest that the mechanisms responsible for the radioprotective action of indometophen may be associated both with its direct effects on the proliferation and differentiation of hemopoietic cellular precursors and with the stimulation of release of growth-differential factors by hemopoietic microenvironmental elements.     

Colony-stimulating factor-induced monocyte survival and differentiation into macrophages in serum-free cultures

The role of mononuclear phagocyte-specific colony-stimulating factor (CSF-1) in human monocyte to macrophage differentiation was investigated. The addition of 1000 U/ml of CSF-1 to serum-free monocyte cultures resulted in monocyte survival comparable to that in cultures containing 5% AB serum, whereas cells in serum- and CSF-1-free medium lost their viability in 3 to 5 days. The requirement for CSF-1 coincided with the time (40 to 64 hr of culture) when the major changes in morphology and biochemical function took place in monocytes undergoing differentiation into macrophages. If CSF-1 was removed from the cultures before this time, death of the monocytes resulted. In cultures containing CSF-1, as in serum containing cultures, the lysosomal enzyme acid phosphatase was enhanced 10- to 20-fold by day 4 to 5. Superoxide production in response to phorbol myristic acetate was maintained in CSF-1 cultured monocytes, but declined with time in monocytes cultured in serum. The expression of monocyte-macrophage antigens p150.95 (LeuM5), OKM1, LeuM3, Fc receptors (32.2), and HLA-DR had increased in CSF-1 containing cultures at day 4. When antigen expression was analyzed at day 2 to 3, when cell size and 90 degrees scatter characteristics were still identical to control serum-free cultures, only p150.95, HLA-DR and FcR expression were enhanced by CSF-1. Low amounts of lipopolysaccharide (0.1 ng/ml) were found to enhance monocyte survival in the absence of added CSF-1. Lipopolysaccharide-containing cultures were found to produce CSF-1 (up to 450 U/ml, as detected by radioimmunoassay). Lipopolysaccharide (1 microgram/ml), however, did not induce enhanced expression of the maturation-related antigens. Based on these observations we conclude that CSF-1 is enhancing human monocyte survival and is involved in the events leading to the differentiation of monocytes into macrophages.     

Suppressive effects of swainsonine and N-butyldeoxynojirimycin on human bone marrow neutrophil maturation

The effects of the N-linked oligosaccharide inhibitors swainsonine and N-butyldeoxynojirimycin (NB-DNJ) on granulopoiesis was investigated using human bone marrow cells in in vitro liquid and agar cultures. The addition of the inhibitors into cultures containing granulocyte colony-stimulating factor (G-CSF) suppressed maturation from myelocytes into mature neutrophils. Swainsonine did not induce apoptosis, but NB-DNJ induced considerable apoptosis, especially in the presence of G-CSF. This result indicated that the decrease of mature neutrophils by swainsonine was not because of cell degeneration. In the case of NB-DNJ, it was thought to be because of both maturation suppression and apoptosis. In a colony-forming unit-granuloid (CFU-G) colony assay, the number of colonies was increased in the presence of the inhibitors, but the morphology of colonies was predominantly compact, or immature. The inhibitors also suppressed the expressions of mRNAs of CCAAT/enhancer binding protein epsilon (C/EBPepsilon) and G-CSF receptor as markers of terminal neutrophil maturation. These findings suggested that the incompleteness of N-linked oligosaccharide leads to the suppression of terminal neutrophil maturation.     

Separate actions of different colony stimulating factors from human placental conditioned medium on human hemopoietic progenitor cell survival and proliferation

More than 20% of human granulocyte-macrophage and eosinophil colony-forming cells survived in agar culture for up to 4 days without the addition of exogenous colony stimulating factors (human placental-conditioned medium, HPCM). Survival was reduced slightly but not significantly, by the removal of adherent cell populations. Significant survival occurred even when only 100 cells enriched for colony-forming cells (CFCs) were cultured per dish. When individual colonies, initiated by stimulation with HPCM for 5 days, were transferred to dishes without HPCM, subsequent proliferation was significantly reduced compared with control cultures containing HPCM. Using the fluorescence-activated cell sorter and the fluoresceinated lectin from Lotus tetragonolobus, two populations of marrow cells were obtained, one enriched for day 7 and the other for day 14 colony-forming cells. Two colony-stimulating factors fractionated from HPLCM (CSF^β and CSF^α) have been shown previously to stimulate the day 7 and day 14 colony-forming cell populations, respectively. Developing clones from cultures initiated with CSFβ died between the fifth and tenth day of culture after transfer to dishes with CSFα or CSFβ or to dishes with no stimulus. Cells in clusters initiated with CSFα proliferated significantly between the fifth and tenth day of culture when transfered to CSFα or CSFβ but not when transfered to dishes with not stimulus. These studies provide further evidence for the existence of two subtypes of human granulocyte-macrophage progenitor cells each under the primary control of a specific regulator and indicate that these two regulators can both act on some developing clones of cells.