Effects of recombinant human H-subunit and L-subunit ferritins on in vitro growth of human granulocyte—monocyte progenitors

We have studied the influence of purified recombinant human H-subunit (rHF, acidic) and L-subunit (rLF, basic) ferritins on in vitro colony formation by normal human granulocyte-macrophage progenitor cells (CFU-GM). Whereas rLF had no significant effect, rHF produced significant decrease in colony formation: mean inhibition of CFU-GM was 38% +/- 13% at 10(-8) M and 22% +/- 13% at 10(-9) M. The inhibitory activity of rHF was lost at 10(-10) M, and was inactivated with a monoclonal antibody recognizing the H subunit, but not with a monoclonal antibody recognizing the L subunit. Although H-type isoferritins were found in normal and leukaemic cells, their concentration in peripheral blood plasma and bone marrow plasma from normal subjects and patients with different haematological disorders including acute leukaemia were 10(-11) M or lower, i.e. levels showing no activity in vitro. We conclude that: (i) acidic H-subunit-rich isoferritins have inhibitory effects on in vitro growth of granulocyte-macrophage progenitors; (ii) levels of these isoferritins in peripheral blood and bone marrow plasma are 2-3 orders of magnitude lower than the effective concentrations in vitro, indicating that these molecules do not behave as circulating regulatory or suppressive factors in vivo.     

Effect of acidic and basic isoferritins on in vitro growth of human granulocyte-monocyte progenitors

Acidic isoferritins have been previously found to be highly potent inhibitors of hematopoietic progenitors at concentrations of 10(-16) to 10(-18) mol/L, and it has been suggested that acidic isoferritin inhibitory activity plays a role in the regulation of normal hematopoiesis and also in the pathogenesis of leukemia. To characterize the ferritin species that affect the in vitro growth of human colony- forming unit-granulocyte-macrophage (CFU-GM), we tested different preparations of basic (L-subunit-rich) and acidic (H-subunit-rich) isoferritins. Three preparations of human liver (basic) ferritin did not show any effects on CFU-GM growth at concentrations up to 10(-9) mol/L, irrespective of the degree of glycosylation. Acidic isoferritins were purified both from HeLa cells and human heart. HeLa cell ferritin did not affect in vitro colony formation. One of two preparations of human heart ferritin, containing 5% glycosylated ferritin, showed a mean inhibition of 26% +/- 8% of the control at 10(-9) mol/L (P less than .02), whereas the other preparation, which contained no glycosylated ferritin, did not show any effect of CFU-GM growth. A preparation enriched for glycosylated acidic isoferritins from human heart was found to produce a mean inhibition of 32% +/- 11% of the control at 10(-9) mol/L (P less than .01), whereas another one was ineffective. A significant part of the inhibitory activity was removed by preincubation with the monoclonal antibody 2A4 directed against human heart ferritin. The present findings indicate that basic isoferritins, ie, the predominant ferritin type in human blood, have no effect on the growth of human CFU-GM, and this is in keeping with indirect clinical evidence. Inhibition of colony formation may be obtained by some preparations of acidic isoferritins that are rich in H subunits and bind to concanavalin A. The mechanism(s) responsible for this are not clear, but the effective concentrations are higher than those found in human blood both under normal conditions and in leukemia. At present, the physiologic significance of the observed inhibitory activity is uncertain.     

Myelodysplastic syndrome (MDS)-associated inhibitory activity on haematopoietic progenitor cells: contribution of monocyte-derived lipid containing macrophages (MDLM)

We studied myelodysplastic syndrome-associated inhibitory activity (MDS-IA), which inhibited colony formation in vitro of normal granulocyte-macrophage progenitors (CFU-GM). When adherent marrow cells were incubated with fetal calf serum for 21-24 d, monocyte-derived lipid containing huge macrophages (MDLM) developed. MDLM from MDS marrow (MDS-MDLMs) and their conditioned medium (MDLM-CM) consistently suppressed the growth of normal CFU-GM colony formation. MDS-IA was active on CFU-GM during the S-phase and relatively resistant to heating. Monoclonal antibody against H subunit (acidic) ferritin and polyclonal antibody against placental ferritin neutralized the inhibitory activity of MDS-MDLMs. In addition, cell lysates of MDS-MDLMs reacted to both monoclonal anti-H subunit ferritin and polyclonal anti-placental ferritin in Western blotting analysis, indicating that the inhibitory activity was predominantly acidic isoferritin. On the other hand, MDLMs obtained from normal bone marrow had a CFU-GM enhancing activity. These results suggest that MDS-MDLMs may be responsible for the suppression of granulopoiesis in patients with MDS and that the suppression may be mediated by soluble factors, notably H subunit isoferritin.     

Deoxycytidine stimulates the in vitro growth of normal CFU-GM and reverses the negative regulatory effects of acidic isoferritin and prostaglandin E1

We have examined the effect of supraphysiologic concentrations of the naturally occurring nucleoside deoxycytidine (dCyd) on the in vitro growth of normal (CFU-GM) and leukemic (L-CFU) myeloid progenitor cells. Bone marrow samples obtained from 34 consecutive patients undergoing routine diagnostic bone marrow aspirations for nonmalignant hematologic disorders exhibited nearly a twofold increment in CFU-GM when continuously cultured in the presence of 10(-4) mol/L dCyd. Higher dCyd concentrations were associated with a smaller degree of enhancement of colony formation. In contrast, the growth of leukemic blast progenitors obtained from patients with acute nonlymphocytic leukemia were not enhanced by any of the dCyd concentrations tested. Coadministration of 10(-3) mol/L tetrahydrouridine (THU), a cytidine deaminase inhibitor, failed to alter the relative inability of dCyd to enhance L-CFU colony growth. The stimulatory effect of dCyd on normal CFU-GM was not mediated by the adherent mononuclear cell population of the marrow, nor was it restricted to the subpopulation of CFU-GM in S phase at the time of initial exposure. Moreover, treatment of normal bone marrow cells with dCyd at concentrations ranging from 10(-6) to 5 X 10(-3) mol/L for 24 hours had only a minor effect on the fraction of CFU-GM in S phase. Coadministration of 10(-4) mol/L dCyd was able to reverse the inhibitory effects of several putative regulators of normal myelopoiesis, including leukemia inhibitory activity (LIA), acidic isoferritins (AIF), and prostaglandin E1 (PGE1). Leukemic myeloblasts exposed to 10(-4) mol/L dCyd exhibited substantial expansion of intracellular pools of dCyd triphosphate (dCTP), demonstrating that inability to metabolize dCyd could not be solely responsible for the absence of growth potentiation in these cells. These studies suggest that supraphysiologic concentrations of dCyd may confer a selective in vitro growth advantage upon normal v leukemic myeloid progenitor cells, and may free the former from the inhibitory effects of several potential negative regulators of myelopoiesis.     

The effect of isoferritins on granulopoiesis

The evidence for a regulatory role of acidic isoferritins on hemopoiesis is not entirely consistent with our knowledge of ferritin biochemistry, and no clear picture of this phenomeonon has emerged. In the present study, we have been unable to confirm a consistent effect of purified heart (acidic), spleen (basic), or serum (glycosylated) isoferritins on CFU-GM colony formation in vitro. Inhibition of colony formation by cell extracts or conditioned media does not relate to the presence of acidic isoferritins, nor is this effect neutralized by a monoclonal antibody to acidic isoferritins. The composition of ferritin preparations previously described as inhibitory to CFU-GM colony growth could not be confirmed, and they were not found to be predominantly acidic in nature. Our data do not support a role for acidic isoferritins as inhibitors of granulopoiesis.     

Abnormalities in myelopoietic regulatory interactions with acidic isoferritins and lactoferrin in mice infected with Friend virus complex: association with altered expression of Ia antigens on effector and responding cells

The regulation of myelopoiesis was evaluated in B6D2F1 mice inoculated with Friend virus complex (spleen focus-forming virus plus helper virus) or helper virus alone by analyzing acidic isoferritin (AIF) and lactoferrin (LF) interactions with target cells. Under normal conditions, AIF suppresses colony and cluster formation by an Ia- antigen-positive cycling subpopulation of mouse granulocyte-macrophage progenitor cells (CFU-GM). Under the same conditions, the release of AIF-inhibitory activity and granulocyte-macrophage colony stimulatory factors (GM-CSF) from an Ia-antigen-positive subpopulation of monocytes and macrophages is suppressed by LF. Within one to two days after inoculation in vivo with Friend virus complex or helper virus, mouse CFU-GM become insensitive in vitro to suppression by purified human AIF as well as crude mouse AIF, and by four days, bone marrow, spleen, and thymus cells of these mice release much greater quantities of AIF- inhibitory activity than the cells from mice injected with control medium. The Friend virus complex itself has no influence in vitro on CFU-GM from normal mice. In addition, the release of AIF-inhibitory activity from bone marrow, spleen, and resident peritoneal cells and the release of GM-CSF from resident peritoneal cells of mice infected with Friend virus complex are not suppressed by LF. The inability of AIF to suppress colony formation by bone marrow and spleen CFU-GM from mice infected with Friend virus complex is associated with the loss of Ia (I-A subregion) antigens from CFU-GM, even though CFU-GM are in cycle. The nonresponsiveness of bone marrow, spleen, and peritoneal cells from these mice to LF suppression of AIF release and the inability of LF to influence GM-CSF release from peritoneal cells is associated with loss of Ia antigens from these cells. The above abnormalities are similar to the defects noted using cells from patients with leukemia. These results suggest that mice infected with Friend virus complex can serve as a model for investigating abnormalities in cell regulation and their relationships to disease progression.     

Myelodysplastic syndrome (MDS)-associated inhibitory activity on haemopoietic progenitor cells

We studied MDS-associated inhibitory activity, which inhibited colony formation in vitro of granulocyte-macrophage progenitors (CFU-GM). Macrophages obtained from MDS bone marrow mononuclear cells (BM-MNC) when pretreated with granulocyte-macrophage colony stimulating factor (GM-CSF) suppressed the growth of normal CFU-GM. These macrophages were designated as 'MDS-derived inhibitory macrophages'. Media conditioned by MDS-derived inhibitory macrophages (MDS-CM) also suppressed the growth of normal CFU-GM. In the MDS-CM, high levels of prostaglandin E2 (PGE2) and ferritin were found. However, MDS-CM did not contain detectable levels of tumour necrosis factor (TNF) or gamma-interferon (gamma-IFN). Antiserum against human placental ferritin and/or against PGE2 blocked the haemopoietic inhibitory activity to some extent. These results suggest that inhibitory macrophages may be responsible for the suppression of granulopoiesis in patients with MDS and that the suppression may be mediated by soluble factors including PGE2 and ferritin.     

Suppressive effects in vivo of purified recombinant human H-subunit (acidic) ferritin on murine myelopoiesis

Purified recombinant human heavy-chain (acidic) ferritin (rHF) was assessed in vivo in mice for effects on the proliferation (percentage of cells in S-phase) and absolute numbers of granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells in the femur and spleen and on the nucleated cells in the marrow, spleen, and blood. rHF significantly decreased cycling rates and absolute numbers of marrow and splenic hematopoietic progenitors and marrow and blood nucleated cellularity. These effects were apparent in BDF1, C3H/Hej and DBA/2 mice and were dose dependent, time related, and reversible. Suppressive effects were noted within three hours for progenitor cell cycling, within 24 hours for progenitor cell numbers, and within 48 hours for circulating neutrophils. Additionally, hematopoietic progenitor cells in DBA/2 mice infected with the polycythemia-inducing strain of the Friend virus complex (FVC-P) were insensitive to the in vivo administration of rHF. These studies demonstrate activity of rHF in vivo on myelopoiesis of normal but not FVC-P-infected mice. Since rHF suppresses hematopoietic progenitor cell proliferation from normal donors in vitro and from normal mice in vitro and in vivo but does not suppress progenitor cells from patients with leukemia in vitro or from mice with FVC-P-infection in vitro or in vivo, rHF may be useful as a candidate adjunct molecule for the protection of normal hematopoietic progenitor cells during chemotherapy.     

Monocyte-macrophage-derived acidic isoferritins: normal feedback regulators of granulocyte-macrophage progenitor cells in vitro

The recent identification of a leukemia-associated inhibitory activity (LIA) against granulocyte-macrophage progenitor cells (CFU-GM) as acidic isoferritins has now led to detection of this activity in normal bone marrow and blood cells. Detection of this activity depends on stimulation of CFU-GM by granulocyte-macrophage colony stimulatory factors (GM-CSF), and some conditioned media (CM) sources of GM-CSF (human placental and monocyte, mouse macrophage and WEHI-3) contained low levels of acidic isoferritin that lowered colony formation. Inactivation or removal of this activity increased the stimulatory capacity of the CM. CM depleted of acidic isoferritins or CM originally devoid of this activity (human GCT, 5637, Mo, lymphocytes: mouse L cells or pokeweed-mitogen-stimulated spleen cells) increased the sensitivity of the assay to detect acidic isoferritin inhibitory activity. This activity was selectively contained and released from normal monocytes and macrophages. Restriction of this activity to mononuclear phagocytes was substantiated, as only continuous cell lines of monocytes and macrophages or lines capable of induction to this lineage contained and released acidic isoferritin inhibitory activity. The cells of origin and target cells of action suggest that acidic isoferritin-inhibitory activity can be considered as a negative feedback regulator, at least in vitro.     

Deoxycytidine preferentially protects normal versus leukemic myeloid progenitor cells from cytosine arabinoside-mediated cytotoxicity

We examined the ability of high concentrations of the naturally occurring nucleoside deoxycytidine (dCyd) to reverse the cytotoxicity of high (eg, greater than or equal to 10(-5) mol/L) concentrations of 1- B-D arabinofuranosylcytosine (Ara-C) toward normal (CFU-GM) and leukemic myeloid progenitor cells (L-CFU). Leukemic myeloblasts from patients with acute nonlymphocytic leukemia (ANLL) and normal human bone marrow mononuclear cells were cultured in soft agar in the continuous presence of 10(-5) to 5 X 10(-5) mol/L of Ara-C together with dCyd (10(-4) to 5 X 10(-3) mol/L). Administration of 10(-5) mol/L of Ara-C alone eradicated colony formation in all samples tested. Coadministration of 10(-3) mol/L of dCyd restored 72.2% of control colony formation for CFU-GM, but only 10.9% for L-CFU. When higher concentrations of Ara-C (eg, 5 X 10(-5) mol/L) were administered, dCyd- mediated protection toward CFU-GM decreased, but remained significantly greater than that observed for L-CFU. Incubation with 10(-3) mol/L of dCyd reduced the 4-hour intracellular accumulation of the triphosphate derivative of Ara-C (Ara-CTP) in both normal and leukemic cells by greater than 98%; under identical conditions, a significant expansion of the intracellular of the triphosphate derivative of dCyd (dCTP) pools was observed in normal bone marrow mononuclear cells but not in leukemic blasts. This finding was associated with a greater reduction in Ara-C DNA incorporation in normal elements. These in vitro studies suggest that dCyd may preferentially protect normal v leukemic myeloid progenitor cells from the lethal actions of high-dose Ara-C.     

Biosynthesis of ferritin subunits from different cell lines of HL-60 human promyelocytic leukaemia cells and the release of acidic isoferritin-inhibitory activity against normal granulocyte-macrophage progenitor cells

Biosynthesis of acidic isoferritins was investigated in human promyelocytic HL-60 cells, characterized by diploid (2C), tetraploid (4C) and mixed diploid–tetraploid (2C–4C) DNA cell lines. The three cell lines were studied for the biosynthesis of ferritin and its subunits and for the release of acidic isoferritin-inhibitory activity against normal CFU-GM before and after addition of DMSO. While the tetraploid and mixed diploid–tetraploid cell lines synthesized more H- ( M _r= 21) than L-subunits ( M _r= 19) after induction, the tetraploid line synthesized more H-subunit before and after induction, compared to the diploid line. The release of acidic isoferritin-inhibitory activity was greater before than after induction in both cell lines, but the tetraploid cell line released more acidic isoferritin-inhibitory activity consistent with its greater production of M _r= 21 subunit. However, after induction no inhibitory activity could be detected from the diploid cells and much less activity was detected with the tetraploid cells, suggesting that differentiation caused a decrease in production of acidic isoferritin-inhibitory activity.     

Activities derived from established human myeloid cell lines reverse the suppression of cell line colony formation by lactoferrin and transferrin

Myeloid cell lines were evaluated for the release of substances needed for colony formation by their own colony-forming cells (CFC) and by other myeloid cell lines. Dialyzed U937 conditioned medium (CM) had no effect on the cloning efficiency of U937 cells, whether or not U937 CFC had been induced for MHC class-II antigens by preincubation of these cells for 72 h with indomethacin and human gamma interferon (HuIFN gamma). Dialyzed U937 CM, however, restored colony formation of HuIFN gamma-induced U937 cells suppressed by lactoferrin (LF) or transferrin (TF). Dialyzed U937 CM did not restore colony formation of U937 cells suppressed by acidic isoferritins (AIF) or prostaglandin E2 (PGE2). Detection of the growth-restoring effects of U937 CM required that U937 CM be prepared in the presence of indomethacin or that the CM be dialyzed to remove inhibitors of U937 colony formation. Dialyzed U937 CM did not inactivate LF. Dialyzed U937 CM did not stimulate or enhance colony formation of normal human bone marrow granulocyte-macrophage (CFU-GM), erythroid (BFU-E), or multipotential (CFU-GEMM) progenitor cells, but did contain potent inhibitory activity against these progenitor cells. HL-60, EM2, EM3, and K562 cells were also evaluated. HL-60-, EM3-, and K562-CFC that were not preincubated with HuIFN gamma did not express MHC class-II antigens, and colony formation by these cells was not influenced by LF, TF, or AIF. Noninduced EM2-CFC constitutively expressed MHC class-II antigens, and colony formation by these cells was suppressed by LF, TF, and AIF. After induction of MHC class-II antigens on HL-60- and EM3-CFC by HuIFN gamma, colony formation by these cells was suppressed by LF, TF, and AIF. Colony formation by HuIFN gamma-induced EM2 cells was more responsive to inhibition by LF, TF, and AIF than was colony formation by noninduced EM2 cells. K562 cells were not induced into a responsive state to LF, TF, or AIF by HuIFN gamma. Dialyzed CM from HL-60, EM2, and EM3 cells contained activities that restored colony formation by their own LF-suppressed CFC. The activities present in dialyzed CM from U937, HL-60, EM2, and EM3 cells may be similar since they could each restore LF-suppressed colony formation of U937, HL-60, EM2, or EM3 cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Progesterone inhibits proliferation of human marrow colony forming cells (CFU-GM) through increased prostaglandin production by marrow macrophages

Progesterone inhibited proliferation of granulocyte/macrophage progenitor cells (CFU-GM) in human bone marrow cultured in methylcellulose; the inhibition ranged from 8% at 3 X 10(-9) M, 22% at 3 X 10(-8) M to 38% at 3 X 10(-7) M progesterone in 14 non-malignant marrows. Depletion of marrow monocytes/macrophages with a monoclonal antibody MO2, abolished the inhibitory effect of progesterone. However, production of colony stimulating activity (CSA) did not appear to be affected, either in PHA stimulated peripheral blood or in bone marrow. Indomethacin, a prostaglandin synthetase inhibitor, greatly reduced the degree of inhibition and the addition of prostaglandin E1 (PGE1) to culture in concentrations as low as 10(-12) M completely removed any additional inhibitory effect of progesterone. PGE1 is known to inhibit only those CFU-GM bearing the Ia antigen; we observed that depletion of Ia positive CFU-GM by either 24 h incubation or by monoclonal (I2) treatment, reduced or abolished progesterone mediated inhibition. This confirms that the mechanism of progesterone inhibition of CFU-GM is mediated through increased PGE production by marrow macrophages, and subsequent inhibition by PGE of Ia positive CFU-GM.     

Identification of leukemia cell-derived inhibitory activity (LIA) in conditioned media from human myeloid leukemic cell line ML-2

Summary Conditioned media from the human myeloid leukemic cell line ML-2 contain a factor that inhibits the entry of normal CFU-GM into S phase of mitotic cycle as measured by the ³H-TdR suicide technique. This factor was detected in conditioned media prepared by incubating 5×10⁶ ML-2 cells/ml or 1×10⁶ ML-2 cells/ml in serum-free RPMI for 5 or 24 hours respectively, and was isolated by ultrafiltration through an XM300 Diaflo membrane followed by chromatography on Sepharose 6B. Ferritin, prepared from human placenta, had the same inhibitory effect on CFU-GM. Antibodies against human placental ferritin completely inactivated the inhibitory effect of both human placental ferritin and the factor released from ML-2 cells. The inhibitory activity produced by the cell-line ML-2 was considered as LIA (leukemia cell-derived inhibitory activity) earlier found in HL-60 cell line and AML and CML cells.     

Effect of recombinant human tumor necrosis factor on the colony growth of human leukemia progenitor cells and normal hematopoietic progenitor cells

The effects of recombinant human tumor necrosis factor (rH-TNF) on the colony growth of human leukemia progenitor cells (L-CFU), granulocyte- macrophage progenitor cells (CFU-GM), and erythroid progenitor cells (BFU-E) were studied. L-CFU was assayed with leukemia cells obtained from patients with acute myelogenous leukemia. CFU-GM and BFU-E were assayed with bone marrow cells obtained from hematologically normal donors and patients with acute leukemia or non-Hodgkin's lymphoma in complete remission. A dose-dependent growth inhibition of L-CFU as well as CFU-GM and BFU-E was observed by rH-TNF at concentrations of 1 to 100 U/mL. The inhibitory effect on L-CFU was significantly greater than that on CFU-GM. No correlation was observed between the inhibitory effect on L-CFU and the number of colonies formed in the cultures without rH-TNF. Preincubation of the progenitor cells in culture medium containing 20% fetal calf serum with up to 1,000 U/mL of rH-TNF for 24 hours did not result in the inhibition of colony growth of L-CFU or CFU- GM. The inhibitory effect of rH-TNF was neutralized by an anti-rH-TNF murine monoclonal antibody.     

The dimer of hemoregulatory peptide (HP5B) stimulates mouse and human myelopoiesis in vitro

A synthetic analogue of a pentapeptide associated with mature granulocytes has been described earlier and shown to suppress myelopoietic colony formation in vitro in concentrations from 10(-13) to 10(-6) M. By oxidation of the peptide, a dimer will rapidly occur by formation of disulfide bridges between cysteine residues. We here demonstrate that this dimer has the opposite effects of the monomer. For both mouse and human granulocyte-macrophage colony-forming units (CFU-GM), a dose-dependent enhancement of colony formation was observed in the dose range 10(-16) to 10(-5) M, where a saturation level was reached above 10(-8) M. At low doses of colony-stimulating activity (CSA) and in the linear stimulating phase, an up to ten times increase of colony formation was seen, whereas at higher doses the effect was less pronounced. Also at the plateau level of CSA stimulation an increased colony yield was seen. All types of colonies were stimulated. The dimer itself had no colony-stimulating factor activity and was not toxic to bone marrow cells in suspension cultures up to 24 h. Upon reduction of the dimer by use of sulfhydryl compounds, inhibitory effects on CFU-GM were restored. The peptide had no effect on the phagocytic process in human granulocytes, including attachment and internalization of bacteria or Zymosan particles. The monomerdimer equilibrium of hemoregulatory peptide may constitute a new mechanism for proliferative regulation of myelopoietic cells.     

Hydrocortisone promotes survival and proliferation of granulocyte-macrophage progenitors via monocytes/macrophages

We have determined the mechanism by which hydrocortisone (Hc) promotes the survival and proliferation of normal human granulocyte-macrophage progenitors (granulocyte-macrophage colony-forming units, CFU-GM). Peripheral blood mononuclear cells were cultured in a liquid system with 0-10.0 microM Hc over 3 weeks. At 7-day intervals 50% of the culture media along with the cells (suspension cells) present in the media were removed and replaced with fresh media. No CFU-GM or very small numbers of CFU-GM were contained in the suspension cells of the 14- and 21-day-old liquid cultures without Hc; CFU-GM were present and increased with increasing concentrations of Hc. The CFU-GM content in suspension cells of 14- and 21-day-old liquid cultures with 1.0 microM Hc was at least threefold higher compared to liquid cultures without Hc. In a double-layer CFU-GM agar culture system, the suspension cells from liquid cultures with 1.0 microM Hc, but not from liquid cultures without Hc, supported CFU-GM proliferation from normal human bone marrow cells. The CFU-GM proliferation-inducing ability was confined to the monocytes/macrophages (Mo). CFU-GM colony inhibitory and stimulatory activities were detected in cell-free media recovered from liquid cultures without Hc, but only colony stimulatory activity was detected in the media from cultures with 1.0 microM Hc. These results indicate that greater than or equal to physiological concentrations of Hc (0.1-1.0 microM) are required for the persistence and proliferation of CFU-GM, and the effect of Hc is mediated through the Mo, probably by inhibiting the production of one or more of the CFU-GM colony inhibitory molecules.     

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.     

Acidic isoferritins (leukemia-associated inhibitory activity) fail to inhibit blast proliferation in acute myelogenous leukemia

Cell-free extracts of bone marrow and blood cells from patients with leukemia contain an inhibitor of normal granulocyte/macrophage progenitor (CFU-GM) proliferation (leukemia-associated inhibitory activity, LIA) identified as acidic isoferritins. A comparison was made of the action of crude LIA prepared from frozen-thawed leukemic blood cells and purified spleen ferritin from a patient with chronic myelogenous leukemia, on the proliferation of blast progenitors from patients with acute myelogenous leukemia (AML), and on the promyelocytic leukemia cell line, HL-60. Crude LIA showed no inhibition of blast progenitor or HL-60 proliferation at low concentrations, but inhibited the proliferation of CFU-GM. At higher concentrations, crude LIA inhibited both blast cells and CFU-GM. Purified spleen ferritin failed to inhibit blast progenitors or HL-60 cells at any concentration tested, but inhibited both 70-day and 14-day CFU-GM. Using the thymidine "suicide" technique, the action of LIA was confirmed as being on CFU-GM in S-phase, but it failed to affect the proliferation of blast cell in S-phase. It is concluded that acidic isoferritins inhibit normal CFU-GM but not blast cells from patients with AML. Acidic isoferritins could confer a proliferative advantage of the leukemic clone over its normal counterparts.