Possible role of gangliosides in the interaction of colony-stimulating factor with granulocyte-macrophage progenitor cells

Our previous studies have shown that preincubation of murine bone marrow (BM) cells with cholera toxin (CT) or with its B subunit inhibited their responsiveness to colony-stimulating factor (CSF). Because ganglioside GM1 is a component of the CT receptor, the present study was undertaken to determine whether gangliosides interact with CSF and therefore might play a role in the binding sites for CSF. Preincubation of CSF with increasing concentrations of bovine-brain mixed gangliosides resulted in decreased numbers of colonies of BM-derived granulocyte-macrophage progenitor cells (CFU-C) in soft agar. The inhibitory effect of the gangliosides could be reduced by increasing the concentrations of CSF. Evidence for direct binding of CSF to gangliosides was obtained by affinity chromatography of CSF on gangliosides-sepharose beads. CSF activity was retained on the beads and could be eluted with 6 M guanidine HCl. Four different individual gangliosides (GM1, GM2, GD1a, GT1b) were tested for their inhibitory effect on CSF-induced clonal growth of CFU-C. GM1 was the most effective with a 50% inhibition (I50) of clonal growth at a concentration of 15 microM. while the other three gangliosides had slight inhibitory activity (I50 at a concentration greater than 100 microM). In addition, preincubation of BM cells with rabbit anti-GM1 antibodies before addition of CSF reduced the clonal growth of CFU-C to 45%. These data indicate that GM1 interacts with CSF and suggest that gangliosides may play a role in the interaction of CSF with CFU-C and that the binding site for CSF on the surface of these cells might either consist of or contain this ganglioside.     

RAS-blocking bisphosphonate zoledronic acid inhibits the abnormal proliferation and differentiation of juvenile myelomonocytic leukemia cells in vitro

Juvenile myelomonocytic leukemia (JMML) is a clonal myeloproliferative/myelodysplastic disorder of early childhood with a poor prognosis. JMML cells are characterized by hypersensitivity to granulocyte-macrophage colony-stimulating factor (GM-CSF) caused by a continuously activated GM-CSF receptor-retrovirus-associated sequence (RAS) signal transduction pathway through various molecular mechanisms, resulting in spontaneous GM colony formation in vitro. Bisphosphonate zoledronic acid (ZOL), a RAS-blocking compound, suppressed colony formation from bone marrow (BM) cells of 8 patients with JMML and 5 healthy control subjects without and with GM-CSF (10 ng/mL), respectively, in a dose-dependent manner in clonal culture. At 10 microM ZOL, however, spontaneous GM colony formation from JMML BM cells decreased to 3%, but the formation of G colonies containing granulocytes, but no macrophages, was enhanced, whereas 40% of GM colonies were retained and G colony formation was not affected in culture of normal BM cells with GM-CSF. In suspension culture, cytochemical and flow cytometric analyses showed that 10 microM ZOL also inhibited spontaneous proliferation and differentiation along monocyte/macrophage lineage of JMML BM cells but not the development of normal BM cells by GM-CSF. The inhibitory effect of ZOL on JMML cells was confirmed at a single-clone level and observed even at 3 microM. The current result offers a novel approach to therapy in JMML.     

Putative involvement of protein kinase C in proliferation of human myeloid progenitor cells

The effects of two different potent inhibitors of protein kinase C, 1- (5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) and staurosporine on human myeloid (CFU-C) and late erythroid progenitor cells (CFU-E) were studied using an in vitro clonal assay. Our objective was to determine whether protein kinase C has a role in signal transduction related to proliferation of these committed progenitor cells. The presence of H-7 or staurosporine led to an inhibition of colony formation stimulated by crude colony-stimulating factor (CSF), interleukin-3 (IL-3), granulocyte-macrophage CSF (GM-CSF), granulocyte CSF (G-CSF), or macrophage CSF (M-CSF) in a dose-dependent manner. N-(2-guanidinoethyl)- 5-isoquinolinesulfonamide (HA-1004), a weaker analog of H-7, did not inhibit proliferation of CFU-C. Neither H-7 nor staurosporine had any effect on CFU-E formation. H-7 and staurosporine dose-dependently inhibited the protein kinase C from K562 cells. The potential of these compounds to inhibit proliferation of CFU-C correlated well with the magnitude of their inhibition of protein kinase C from K562 cells. The inhibition of proliferation of CFU-C appears to relate to the potential of these compounds to inhibit protein kinase C. Thus, activation of protein kinase C is presumably involved in the proliferation of CFU-C, and the regulatory system of CFU-E appears to differ from that of CFU-C.     

In vitro hepatitis B virus suppression of erythropoiesis is dependent on the multiplicity of infection and is reversible with anti-HBs antibodies

Exposure of human bone marrow mononuclear cells to hepatitis B virus results in the suppression of the in vitro growth of several hematopoietic progenitor cells. We studied the degree of inhibition of erythroid progenitor cells that results as a function of the time of exposure of mononuclear cells to hepatitis B virus and the ratio of virus to mononuclear cells, the multiplicity of infection. With an overnight incubation of mononuclear cells with hepatitis B virus-containing sera, a multiplicity of infection of greater than one virus per mononuclear cell is required to observe significant inhibition of erythroid colony formation. This multiplicity of infection effect is also observed with purified Dane particles. Exposure of mononuclear cells to an increasing number of Dane particles results in a dose-dependent suppression of erythroid colony formation with significant inhibition observed with a multiplicity of infection of virus to mononuclear cells as low as 5:1. Murine monoclonal antibodies to HBsAg completely neutralize the hepatitis B virus-mediated inhibition of CFU-E while control antibodies do not. Purified HBsAg has no effect on colony formation. In conclusion, the hepatitis B virus-mediated inhibition of erythrogenesis in vitro provides a basis for understanding the bone marrow depression observed during hepatitis B virus infections and may provide an in vitro model for examining hepatitis B virus infection.     

Direct growth suppression of myeloid bone marrow progenitor cells but not cord blood progenitors by human cytomegalovirus in vitro

Recently, considerable interest has arisen as to use cord blood (CB) as a source of hematopoietic stem cells for allogenic transplantation when bone marrow (BM) from a familial HLA-matched donor is not available. Because human cytomegalovirus (HCMV) has been shown to inhibit the proliferation of BM progenitors in vitro, it was important to examine whether similar effect could be observed in HCMV-infected CB cells. Therefore, the effect of HCMV challenge on the proliferation of myeloid progenitors from BM and CB was compared using both mononuclear cells (MNC) and purified CD34+ cells. A clinical isolate of HCMV inhibited the colony formation of myeloid BM progenitors responsive to granulocyte-macrophage colony-stimulating factor (CSF), granulocyte- CSF, macrophage-CSF, interleukin-3 (IL-3) and the combination of IL-3 and stem cell factor (SCF). In contrast, colony growth of CB progenitors was not affected. In addition, HCMV inhibited directly the growth of purified BM CD34+ cells responsive to IL-3 and SCF in single cell assay by 40%, wheras the growth of CD34+ progenitors obtained from CB was not suppressed. The HCMV lower matrix structural protein pp65 and HCMV DNA were detected in both CB and BM CD34+ cells after in vitro challenge. However, neither immediate early (IE)-mRNA nor IE proteins were observed in infected cells. Cell cyclus examination of BM and CB CD34+ cells revealed that 25.7% of BM progenitors were in S + G2/ M phase wheras only 10.7% of the CB progenitors. Thus, a clinical isolate of HCMV directly inhibited the proliferation of myeloid BM progenitors in vitro wheras CB progenitors were not affected. This difference in the susceptibility of CB and BM cells to HCMV may partly be caused by the slow cycling rate of naive CB progenitors compared to BM progenitors at the time of infection.     

Elaboration of granulocyte-macrophage colony-stimulating factor by human tumor cell lines and normal urothelium

A total of 72 cell conditioned media (CCM) were screened for their ability to stimulate colony formation by human granulopoietic progenitor cells. Granulocyte-macrophage (GM) colony-stimulating factor(s) (CSF) were found in CCM of nine tumor cell lines, two primary urinary bladder tumors, and three epithelial cell cultures of normal urinary tract. The most active medium came from urinary bladder carcinoma cell line 5637. CSF released by the 5637 cell line induced dose-dependent GM colony formation from human fetal, normal adult, and CML bone marrow (BM) and from mouse BM. Human fetal and normal adult BM formed more colonies when stimulated with 5637 CCM than with peripheral blood leukocyte (PBL) feeder layers, while CML BM produced more colonies with PBL feeder layers. CCM from 5637 was more active in stimulating GM colony formation than human placenta conditioned medium (PCM) and PHA-LCM. 5637 CCM produced in serum-free hormone-supplemented medium was nearly equipotent and can serve as suitable starting material for purification.     

Selective inhibition of the proliferation of various murine hemopoietic progenitor cells by cholera toxin

We have studied in detail the effects of cholera toxin (CT), its pentameric B-chain subunit (toxoid) and the A-promoter chain on the differentiation of hemopoietic progenitor cells. Murine marrow cells were treated either with CT or its subunits. After stimulation with either the multilineage growth factor (multi-CSF; also called interleukin 3 or HCGF) or other hemopoietic regulators (colony-stimulating factors, or CSF), the clones grown in semisolid collagen cultures were scored in situ. Pluripotent stem cells (CFU-S) and multilineage (mixed CFU) or lineage-restricted progenitors (CFU-c) were estimated. We found that CT sensitivity is gradually gained by cells through the stepwise differentiation processes (i.e., CFU-S less than multilineage CFC less than committed CFC less than maturing cells). CT also has a selective, dose-dependent inhibitory effect (1 microM to 1 pM) on hemopoietic lineages (basophil-mast cells less than megakaryocytes less than neutrophils less than monomacrophages). These phenomena were obvious when the clonal growth was supported by multi-CSF but, interestingly, were not observed when lineage-restricted CSF were used. They furnished additional evidence that multi-CSF activates cells in a specific manner. This growth factor involved in progenitor cell self-renewal control may contribute to maintaining, on maturing cells, characteristics that are normally the attributes of progenitors.     

Sensitivity of human acute myeloid leukaemia to diphtheria toxin-GM-CSF fusion protein

The potential to selectively eliminate acute myeloid leukaemia (AML) cells with the GM-CSF-diphtheria toxin fusion protein (DT-GM-CSF) was studied under conditions of autonomous proliferation in vitro with no growth factors (GFs) added and after growth stimulation with a mixture of human (hu)G-CSF, huIL-3 and huSCF. DNA synthesis was maximally inhibited after 48 h exposure to DT-GM-CSF. Cell viability and AML colony forming ability in vitro were reduced. 18/22 samples were found to be sensitive to DT-GM-CSF, with 50% inhibition of DNA synthesis (ID₅₀) at concentrations ranging from 0.1 to 16 ng/ml, and four samples were minimally or not sensitive to DT-GM-CSF (ID₅₀  99 ng/ml). From the 15 samples which showed autonomous proliferation, 13 were sensitive to inhibition of proliferation by DT-GM-CSF. The level of GM-CSF receptor (GM-CSFR) expression was determined by flow cytometry after labelling with specific antibodies for the alpha and beta subunits. Although the toxicity to DT-GM-CSF was specifically mediated by the GM-CSFR, no correlation was found between the level of expression of the GM-CSFR alpha or beta subunit and the sensitivity for DT-GM-CSF. These in vitro studies show that the DT-GM-CSF fusion protein can be used for specifically targeting and eliminating leukaemic cells in the majority of AML cases.     

Characterization of possible receptor of colony-promoting activity (CPA) by comparison with that of colony-stimulating factor (CSF)

Supernatants of long-term mouse bone marrow cell cultures contain colony-promoting activity (CPA). CPA itself does not stimulate colony formation of granulocyte-macrophage progenitor cells (GM-CFC) when added to a semisolid agar culture of bone marrow cells, but augments colony formation in the presence of colony-stimulating factor (CSF). The CPA-responsive cells are postulated to be pre-GM-CFC, a cell compartment younger than GM-CFC. In absorption experiments, CSF-coated bone marrow cells failed to absorb CPA, whereas normal bone marrow cells absorbed the activity. Cholera toxin (CT) is known to inhibit GM-CFC colony formation of bone marrow cells, probably by binding to GM-CFC-receptors [8, 9]. In the present studies, preincubation of the cells with CPA, before exposure to CT, reduced the suppressive effect of CT on GM-CFC colony formation. CT also inhibited pre-GM-CFC colony formation. Such suppressive effects, however, were reduced by preincubation of the cells not only with CPA but also with CSF. These results suggest that CSF and CPA might share the same receptors. It may also suggest that CT does not bind to specific sites. A difference in the susceptibility of GM-CFC and pre-GM-CFC to suppression by CT was also observed. Incubation of the cells with a low concentration of CT resulted in the substantial decrease of the number of GM-CFC, whereas the number of pre-GM-CFC remained high. Therefore, it seems that CPA shares some but not all of the CSF receptors.     

Increased expression of Fas antigen on bone marrow CD34+ cells of patients with aplastic anaemia

Summary Fas antigen, a receptor molecule that mediates signals for programmed cell death, is involved in T-cell-mediated killing of malignant, virus-infected or allogeneic target cells. Interferon- γ (IFN- γ ) and tumour necrosis factored (TNF- α ), potent inhibitors of haemopoiesis, enhance Fas receptor expression on bone marrow (BM) CD34⁺ cells, and both cytokines render haemopoietic progenitor cells susceptible to Fas-mediated inhibition of colony formation due to the induction of apoptosis. Haemopoietic suppression in aplastic anaemia (AA) has been associated with aberrant IFN- γ , increased TNF- β expression, and elevated numbers of activated cytotoxic T-cells in marrow. We have now examined Fas antigen expression in fresh AA BM samples. In normal individuals few CD34⁺ cells expressed Fas antigen and normal marrow cells had low sensitivity to Fas-mediated inhibition of colony formation. In contrast, in early AA, BM CD34⁺ cells showed markedly increased percentages of Fas receptor-expressing CD34⁺ cells, which correlated with increased sensitivity of AA marrow cells to anti-Fas antibody-mediated inhibition of colony formation. The proportion of Fas antigen-bearing cells was lower in recovered patients'BM. Fas antigen was also detected in the marrow of some patients with myelodysplasia, especially the hypocellular variant. These results are consistent with the hypothesis that AA CD34⁺ cells, probably including haemopoietic progenitor cells, express high levels of Fas receptor due to in vivo exposure to IFN- γ and/or TNF-α and are suitable targets for T-cell-mediated killing. Our results suggest that the Fas receptor/Fas ligand system are involved in the pathophysiology of BM failure.     

Inhibition of human bone marrow and myeloid progenitors by interleukin 2-activated lymphocytes

We have shown that unstimulated and interleukin 2 (IL-2)-activated peripheral blood lymphocytes from both normal donors and cancer patients in remission significantly inhibited the proliferation and granulocyte-macrophage colony formation (granulocyte-macrophage colony-forming cells, GM-CFC) of autologous and allogeneic bone marrow (BM). The inhibition was mediated primarily by CD5+ T cells, although lower levels of inhibition were also displayed by CD56+, CD5- lymphocytes, most of which were CD16-. The CD5+ lymphocytes were also the major effectors responsible for lysis of BM. Inhibition of BM proliferation and GM-CFC colony formation was not dependent on proliferation of the effector cells or cell-to-cell contact, because it was also mediated by a soluble factor produced by IL-2-activated lymphocytes. The relevance of these findings to future approaches to leukemia treatment is discussed.     

Induction of colony-stimulating factor receptor expression on hematopoietic progenitor cells: proposed mechanism for growth factor synergism.

In many cells systems, the cellular interaction between two or more humoral factors leads to a synergistic response in terms of cellular growth and function. In particular, the growth and differentiation of hematopoietic progenitor cells involves numerous synergistic interactions between colony-stimulating factors (CSFs) that individually stimulate hematopoiesis (granulocyte-CSF, granulocyte-macrophage-CSF, and interleukin-3 [IL-3]), as well as between these factors and other cytokines that individually have no proliferative effect on progenitor cell growth (IL-1 and IL-6). The present study investigated whether hematopoietic growth factor (HGF) synergy could be mediated by upregulation of CSF receptors. Synergistic effects on bone marrow (BM) progenitor cell colony formation, regardless of the combination of factors used, were consistently preceded by increased CSF receptor expression on highly enriched BM progenitor cells, but not on unfractionated BM cells. Induction of CSF receptors preceded detectable differentiation and did not require cell division because nocodazole, an inhibitor of mitosis, blocked CSF-mediated cell proliferation, but not receptor upregulation. Furthermore, combinations of cytokines that did not synergize also failed to affect the level of CSF receptors on BM progenitors. These results have led us to propose a model for HGF synergy whereby one mechanism of action the investigated synergistic cytokines might be the ability to induce increased expression of CSF receptors.     

Mechanism of ranitidine associated anemia

Ranitidine, an H2 receptor antagonist, has been associated with hematotoxicity. The mechanism(s) underlying the toxicity is not well understood. The authors studied the mechanism of anemia in a patient with ranitidine associated anemia and thrombocytopenia. Clinical evaluation suggested drug-induced Coombs' positive reticulocytopenic hemolysis. In vitro, with the patient off ranitidine, the authors were able to induce Coombs' positivity by incubating patient's red cells with ranitidine and his serum. This process was inhibited by prior exposure of his red cells to histamine. In vitro studies using clonal assays for hematopoietic progenitors revealed that while the patient's serum or ranitidine alone did not affect the patient's or normal bone marrow hematopoiesis, the simultaneous presence of both agents significantly suppressed both patient's and normal erythroid progenitor (BFU-E) colony development. This suppressive effect was prevented by the prior exposure of marrow to histamine and was not observed when the patient's serum was heat inactivated. These studies suggest that the anemia may have resulted from complement-dependent autoimmune destruction/inhibition of progenitor/mature erythroid cells by a process critically dependent on the presence of ranitidine and possibly acting at or near the histamine receptor.     

Modulation of enterotoxin binding and function in vitro and in vivo

The use of the nontoxic B subunits of cholera and Escherichia coli enterotoxins in vitro and in vivo led to a decrease in toxin binding to target cells and a decrease in toxin-induced effects (i.e., morphological effects, adenylate cyclase activation, and fluid secretion). The reduction in toxin binding involves a process of down-regulation of cellular receptors for the toxin and not toxin occupancy of receptors. The extent of inhibition was dependent on the amount of B subunit used and on the duration of time after its use. Thus, in vivo exposure to a single bolus of B subunit was sufficient to block toxin binding and activity for up to 18 h. Because the B subunit binds extensively to the esophagus and the stomach, peroral administration will require a preparation that allows the subunit to reach the small bowel in a protected form. Our data provide a rationale for using B subunit therapy for short-term protection against the effects of enterotoxins, before the development of an immune response.     

Normal colony stimulating factor (CSF) production by bone marrow stromal cells and abnormal granulopoiesis with decreased CFUc in S1/S1d mice.

The concentration of CFUC and the production of stromal-derived CSF in the femora of S1/S1d mice were determined. There was a lower concentration CFUC and a smaller total number of nucleated cells in the femoral marrow of WCB6. S1/S1d (S1/S1d) mice than in WCB6. +/+/(+/+) mice or in C57B1/6. +/+ (C57Bl) mice. On the other hand stromal-derived CSF production by femora from S1/S1d mice did not differ significantly from that of +/+'s. These observations indicate that the microenvironmental defect of S1/S1d mice results in decreased growth of granulocytic precursors as well as those of erythroid and megakaryocytic cells. This is consistent with the reported decrease in multipotential stem cell proliferation. Stromal cell derived CSF production was normal and could not be implicated in the decreased production of granulocytic precursors.     

Cholera toxin enhances factor-dependent colony growth of murine mast cell progenitors

Mast cell colonies were observed when mouse spleen or bone marrow cells were cultured in the presence of medium conditioned by concanavalin-A-stimulated spleen cells, indicating that the medium contains the factor(s) necessary for the formation of these colonies. This factor-dependent colony growth of mast cell progenitors was enhanced by cholera toxin and prostaglandin E, which act on cellular growth mainly by elevating the intracellular cyclic-AMP level. The effect of the toxin was neutralized by preincubation of the toxin with GM1 ganglioside, the receptor substance for cholera toxin, suggesting that cholera toxin exerts its action through GM1 gangliosides present on mast cell progenitors. The toxin B subunit, which binds to GM1 ganglioside but does not elevate intracellular cyclic AMP level, did not affect the colony growth of mast cell progenitors. From these results, it is suggested that intracellular cyclic AMP levels may be involved in colony growth of mast cell progenitors.     

Generation of CFUC suppressor T cells in vitro: VII. T derived colony inhibitory activity (Td/CIA) has no suppressor effect on in vitro immunoglobulin production

T derived colony inhibitory activity (Td/CIA) was obtained from unstimulated T cells from aplastic anemia patients (SAA), or from PWM primed normal T cells. Td/CIA suppressed CFUC growth of normal allogeneic marrow to less than 30% of expected growth. Td/CIA was then added to normal peripheral blood T and B cells, primed with PWM, to test whether it would interfere with in vitro immunoglobulin (Ig) production. When Td/CIA from normal T cells was added to cultures of T + B cells + PWM there was a 2-2.1-fold increase in Ig production. Similarly the addition of Td/CIA from SAA patients also resulted in a 1.4 up to 166-fold increase in Ig production. These results indicate that either (a) the targets for Td/CIA are expressed on hemopoietic but not on T and B cells, or (b) that Td/CIA inactivates an accessory cell which is essential for CFUC growth but not for the PWM driven in vitro B cell differentiation system.     

In vitro regulation of human hematopoiesis by natural killer cells: analysis at a clonal level

We studied the effects of a series of well-characterized clones of human natural killer (NK) cells on the proliferation of highly purified normal marrow hematopoietic progenitor cells. Individual NK clones suppressed granulocyte, monocyte, erythroid, or mixed colony formation in a heterogeneous but clonally stable manner. Inhibition of colony growth required a period of close cell contact between NK cell and progenitor cell with maximum inhibition occurring after 8 to 18 hours of preincubation time. The mechanism of killing was at least partially humoral, however, as cell-free supernatants generated by NK clones "activated" by contact with a target cell also inhibited progenitor cell growth. One of the possible humoral mediators was identified as gamma-interferon by studies with specific neutralizing monoclonal antibodies. These results show that clonal NK lines can be further activated by coming in contact with hematopoietic progenitor cells, resulting in substantial inhibition of colony formation in vitro.     

Fas antigen expression on CD34+ human marrow cells is induced by interferon gamma and tumor necrosis factor alpha and potentiates cytokine-mediated hematopoietic suppression in vitro

Activation of Fas antigen, a cell surface receptor molecule, by its ligand results in transduction of a signal for cell death. The Fas system has been implicated in target cell recognition, clonal development of immune effector cells, and termination of the cellular immune response. Fas antigen expression on lymphocytes is regulated by interferon gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha), cytokines that also have inhibitory effects on hematopoiesis. We investigated Fas antigen expression on human marrow cells and the effects of Fas activation on hematopoiesis in vitro. Freshly isolated immature hematopoietic cells, as defined by the CD34 marker, did not express Fas antigen at levels detectable by fluorescent staining. CD34+ cells, which include progenitors and stem cells, showed low levels of Fas expression in culture, even in the presence of growth factors. Stimulation by TNF alpha and IFN gamma markedly increased Fas antigen expression on CD34+ cells. Anti-Fas antibody, which mimics the action of the putative ligand, enhanced IFN gamma- and TNF alpha-mediated suppression of colony formation by bone marrow (BM) in a dose-dependent manner. This effect did not require the presence of accessory cells. Colony formation from mature (CD34+ CD38+) and immature (CD34+ CD38-) progenitor cells and long-term culture initiating cells were susceptible to the inhibitory action of anti-Fas antibody in the presence of IFN gamma and TNF alpha. Apoptosis assays performed on total BM cells and CD34+ cells showed that anti-Fas antibody induced programmed cell death of CD34+ BM cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Humoral Function of Acute Leukaemic Blasts

Leukaemic blasts from 8 patients with different forms of acute leukaemia were investigated for their capability to produce colony stimulating factor. A second point of investigation was to detect inhibitory activity from the same blasts. The agar technique in its double layer modification was used with bone marrow from C57 B1 mice as target cell population. No single pattern of colony growth was observed. CSF production was absent or very low, when compared with CSF from normal mononuclear cells. However, blasts from 4 patients disclosed inhibition of colony growth. No certain relationship existed between the cytochemic type of leukaemia and the production of either activator or inhibitor of in vitro colony formation.