The effect of 1,25-dihydroxyvitamin D3 on hematopoiesis in long-term human bone marrow cultures

The modulatory effect of 1,25-dihydroxyvitamin D3 (vit D) on the growth of myeloid progenitors and on the composition of the stromal layer in human bone marrow long-term cultures was studied. Vit D (2 X 10(-8) M) caused an enhancement in myeloid progenitor cell (CFU-C) growth in the nonadherent and adherent layers during the entire 5-week incubation period. The vitamin did not alter the differentiation pattern of CFU-C (monocyte-macrophage progenitors CFU-M, granulocytic progenitors CFU-G, or monocyte-granulocyte progenitors CFU-GM). Vit D caused a marked increase in the percentage of lipid-containing cells in the adherent layer and an increase in the number of cells that specifically bound My4 monoclonal antibody (McAb), that reacted positively to fluoride-sensitive alpha-naphthyl acetate esterase, and that phagocytosed Candida albicans (CA). Concentrated supernatants harvested from control cultures showed significant levels of myeloid colony stimulating factor (CSF) activity. The addition of vit D to cultures for 5 weeks did not alter CSF levels. These results suggest that vit D may play a role in hematopoiesis by acting directly on the progenitor cells or via the stromal cell production of stimulatory factor(s).     

Divergent regulation of 1,25-dihydroxyvitamin D3 on human bone marrow osteoclastogenesis and myelopoiesis

The physiologically active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) has influence over osteoclastogenesis and myelopoiesis, but the regulational mechanism is not well-defined. In this report, formation of osteoclast-like (OCL) cells from primitive myeloid colony-forming cells (PM-CFC) as mediated by 1,25(OH)2D3 was examined. Our results present in this report clearly show that 1,25(OH)2D3 dose-dependently stimulated OCL cell formation when added to suspension cultures of individually replated PM-CFC colonies. Marrow cells were plated with either granulocyte-macrophage colony-stimulating factor (GM-CSF) or the human bladder carcinoma cell line 5637 conditioned medium (5637 CM) as the source of colony-stimulating activity. The 1,25(OH)2D3 effect of osteoclast differentiation was associated with a concomitant decrease in clonogenic growth of myelopoietic progenitors in response to colony-stimulating activity. Secondly, the effect of adding the known stimulator of hematopoiesis, interleukin-1beta (IL-1beta) and/or 1,25(OH)2D3 on human myeloid colony growth was assessed. IL-1beta enhanced the formation of primitive myeloid colonies in response to GM-CSF by 160%. On the other hand, 1,25(OH)2D3 dose-dependently inhibited both GM-CSF- and 5637 CM-driven myeloid colony formation by as much as 90% at 100 nM. Addition of IL-1beta to GM-CSF-stimulated cultures dampened the inhibitory effect of 1,25(OH)2D3. The inhibition of myeloid clonogenic growth by 1,25(OH)2D3 was almost abolished (89%) by simultaneously adding anti-tumor necrosis factor-alpha monoclonal antibody (anti-TNF-alpha MoAb) to the culture medium. These results collectively suggest divergent roles for 1,25(OH)2D3 in osteoclastogenesis and myelopoiesis, promoting the differentiation of OCL cells from primitive myeloid cells but inhibiting the proliferation of later myeloid progenitor cells. This inhibition of myeloid progenitors may be mediated by TNF-alpha.     

The suppressive effects of recombinant human tumor necrosis factor-alpha on normal and malignant myelopoiesis: synergism with interferon-gamma

The modulation of growth of normal and leukemic myeloid progenitor cells in soft agar cultures by recombinant human tumor necrosis factor-alpha (TNF alpha) and recombinant human interferon-gamma (IFN gamma) was investigated. TNF alpha inhibited colony formation of all colony types representing different maturational stages of normal progenitor cells committed to the myeloid lineage with different orders of sensitivity. Blast-type colonies derived from patients with acute myelogenous leukemia were more sensitive to TNF alpha inhibition than progenitor cells purified from normal bone marrow or bone marrow from patients with stable-phase chronic myelogenous leukemia. The response of most colony types to IFN gamma was poor. However, when IFN gamma was administered together with TNF alpha, synergistically enhanced antiproliferative effects were detected in all colony types tested. The antiproliferative action of IFN gamma on myelopoiesis was enhanced in culture by the presence of autologous monocytes, presumedly by inducing endogenous production of TNF alpha. However, TNF alpha seemed to act directly on the progenitor cells themselves to suppress their clonal growth, rather than involving accessory marrow elements such as monocytes and/or T lymphocytes.     

1,25-Dihydroxyvitamin D3 inhibits the clonogenic growth of transformed cells via its receptor

Anchorage-independent growth in soft agar is a unique property of transformed cells which is known to be correlated with tumorigenicity. We report here that 1,25-dihydroxyvitamin D3 suppresses colony formation by a number of cultured cancer cell lines in soft agar in a dose dependent manner with an ID50 of 5-7 X 10(-10) M. This effect is also achieved with analogues of 1,25-dihydroxyvitamin D3 in accordance with their binding affinity for the hormone's receptor. Only cells with 1,25-dihydroxyvitamin D3 receptor protein are inhibited in their colony formation by vitamin D analogs indicating that the hormone receptor complex may be integrally involved in the in vitro suppression of the anchorage-independent phenotype.     

The effect of natural killer cells on the development of syngeneic hematopoietic progenitors

To determine whether natural killer (NK) cells are involved in the regulation of hematopoiesis, well-characterized, cell sorter-purified NK cells were incubated with syngeneic bone marrow, and the effect of this interaction on the development of various hematopoietic progenitors was assessed. NK cells were obtained from the peritoneal exudates of CBA/J mice after i.p. infection with live Listeria monocytogenes (LM). These NK cells were nylon wool-nonadherent and were purified by using M1/70, a rat anti-murine macrophage monoclonal antibody, and a fluorescence-activated cell sorter (FACS). Syngeneic bone marrow was incubated overnight with these M1/70-purified NK cells. The cells were then assayed in vitro to determine the effect on the colony formation of the following hematopoietic progenitor cells: the myeloid progenitor that produces mixed granulocyte/macrophage colonies (CFU-G/M), the myeloid progenitor that is committed to macrophage differentiation (CFU-M), and the early erythroid progenitor that is known as the burst-forming unit-erythroid (BFU-E). The marrow cells, after incubation with NK cells, were also injected into lethally irradiated syngeneic recipients to assay for the splenic colony formation capacity of the trilineage myeloid stem cell (CFU-S). Although the formation of BFU-E-, CFU-G/M-, and CFU-M-derived colonies was not adversely affected by the exposure of syngeneic bone marrow to purified NK cells, there was a dramatic decrease in the number of CFU-S-derived colonies. Incubation with NK-depleted cells did not result in an inhibition of colony formation by the CFU-S. Mixing experiments showed that the M1/70-labeled NK cells exerted their effect directly on the CFU-S and not on any accessory cells. The effect of the NK cells on colony formation by the CFU-S could be blocked competitively and selectively by the addition, before incubation, of a classic murine NK tumor target, Yac-1. Another tumor line (WTS) that is poorly recognized by NK cells was less effective in blocking the inhibitory effect of NK cells on CFU-S. The demonstration that purified NK cells can selectively inhibit the development of the tripotential CFU-S may point to the importance of NK cells in the regulation of hematopoiesis, in the development of some types of marrow dysfunction, and in the failure of engraftment of transplanted bone marrow.     

THE IN VITRO DIFFERENTIATION OF DENSITY SUB-POPULATIONS OF COLONY-FORMING CELLS UNDER THE INFLUENCE OF DIFFERENT TYPES OF COLONY-STIMULATING FACTOR

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU-c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density sub-populations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony-stimulating factor (CSF) from mouse lung conditioned medium CSF_MLCM), post-endotoxin mouse serum (CSF_ES) and from human urine (CSF_Hu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU-c was dependent on the type of CSF. Functional heterogeneity was found among CFU-c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macro-phage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU-c.     

Comparative studies of the granulopoietic enhancing effects of biosynthetic human insulin-like growth factors I and II

The effect of biosynthetic human insulin-like growth factor I (IGF-I) and IGF-II on the in vitro growth of human marrow myeloid progenitors in the presence of recombinant human granulocyte colony stimulating factor (rhG-CSF), granulocyte-macrophage CSF (rhGM-CSF), or interleukin-3 (rhIL-3), was investigated. IGF-I and IGF-II similarly enhanced the growth of myeloid progenitors in cultures stimulated with any of the above hemopoietic regulators. Analysis of colony composition showed an increase in the numbers of granulocyte colonies, but no alteration in the numbers of macrophage or granulocyte/macrophage colonies. IGF-I induced an increase of 62 ± 16%, 84 ± 13%, and 107 ± 18% in granulocyte colony numbers in the presence of G-CSF, GM-CSF, or IL-3, respectively. The values for IGF-II were 66 ± 13%, 96 ± 12%, and 91 ± 12%. Similar enhancement of myeloid colony formation by both peptides was also detected in G-CSF and GM-CSF-stimulated cultures of marrow cells that had been depleted of accessory cells, while neither peptide exerted any effect in the presence of IL-3 in such cultures. The growth-promoting effects of IGF-I and IGF-II were completely abrogated by monoclonal antibodies directed against the IGF-I (Type I) membrane receptor. IGF-I and IGF-II thus appear to exert their effects on human marrow myeloid progenitors via a direct mechanism involving the Type I receptor. © 1993 Wiley-Liss, Inc.     

The in vitro differentiation of density sub-populations of colony-forming cells under the influence of different types of colony-stimulating factor.

The in vitro proliferation and differentiation of myeloid progenitor cells (CFU-c) in agar culture from CBA/Ca mouse bone marrow cells was studied. Density subpopulations of marrow cells were obtained by equilibrium centrifugation in continuous albumin density gradients. The formation of colonies of granulocytes and/or macrophages was studied under the influence of three types of colony-stimulating factor (CSF) from mouse lung conditioned medium CSFMLCM), post-endotoxin mouse serum (CSFES) and from human urine (CSFHu). The effect of the sulphydryl reagent mercaptoethanol on colony development was also examined. The density distribution of CFU-c was dependent on the type of CSF. Functional heterogeneity was found among CFU-c with partial discrimination between progenitor cells forming pure granulocytic colonies and those forming pure macrophage colonies. Mercaptoethanol increased colony incidence but had no apparent effect on colony morphology or the density distribution of CFU-c.     

GROWTH OF MOUSE AND HUMAN BONE MARROW IN DIFFUSION CHAMBERS IN MICE

Both murine and human bone marrow cells were cultured in plasma clots which were formed inside diffusion chambers implanted into cyclophosphamide- and saline-treated mice. After an initial fall, the number of mouse bone marrow cells and numbers of mouse myeloid stem cells (CFU-C) and agar cluster-forming units rose faster in the cyclophosphamide-treated animals. These hosts also favored formation of myeloid (CFU-D-G) and erythroid (CFU-D-E) colonies and myeloid clusters in the plasma clot. The number and growth rate of mouse CFU-D-G were higher than those of CFU-C from the same marrow population. These observations suggest the existence of humoral factors stimulating granulocyte progenitor cell replication and differentiation. At its best the increment of CFU-D-E number was equivalent to that caused by a single 0·1 unit erythropoietin dose. Culture of normal human marrow cells resulted in colonies in the plasma clot containing only granulocytes and macrophages. Cyclophosphamide-treated host animals were essential for human CFU-D-G development. Plating efficiency for human marrow myeloid colonies was better in the conventional in vitro agar cultures than in diffusion chambers.     

Murine and human hematopoietic colony formation: a possible regulatory role for intracellular histamine

Increasing number of data suggests that locally produced histamine is involved in regulation of hematopoiesis. In this study the granulocyte/macrophage (CFU-GM) colony formation by normal murine or human bone marrow cells, leukaemic colony formation (CFU-L) by a murine leukemia cell line (WEHI 3B), and colony formation by bone marrow cells from patients with chronic myeloid leukemia (CML) have been examined. We detected mRNA and protein expression of histidine decarboxylase (HDC), the only enzyme responsible for histamine synthesis both in normal bone marrow progenitor cells and in leukaemic progenitors. The significance of in situ generated histamine was shown on colony formation by inhibitory action of alphaFMH (blocking HDC activity, i.e. de novo histamine formation) and by N,N-diethyl-2-[4-(phenylmethyl)phenoxy]-ethanamine-HCl (DPPE) disturbing the interference of histamine with intracellular binding sites. These data provide further confirmation of the role of histamine in development and colony formation of bone marrow derived cells.     

Granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D3-induced monocytic differentiation in murine bone marrow cell cultures.

In a series of studies, we have reported that 1,25-dihydroxyvitamin D (3), a known stimulator of monocytic differentiation, primes bone marrow progenitor cells or promyelocytic HL-60 cells to the actions of several factors involved in both monocytic and granulocytic differentiation. In the present study, we have further examined the combinational effects of 1,25-dihydroxyvitamin D (3) and the other inducer of granulopoiesis, granulocyte colony-stimulating factor, on non-fractionated native murine bone-marrow cell culture. Over 6 days of treatment, human granulocyte colony-stimulating factor sustained cell viability, increased the size of small rounded non-adherent cells, and induced granulocytic differentiation, while 1,25-dihydroxyvitamin D (3) decreased cell viability, promoted the development of large adherent flattened cells, and upregulated some monocytic differentiation markers. Combining these two factors over 6 days synergistically upregulated phagocyte activity, membrane-bound interleukin-1alpha, NAD(P)H oxidase, monocytic Mac-1, and non-specific esterase. Similar effects were observed in successive treatment with granulocyte colony-stimulating factor followed by 1,25-dihydroxyvitamin D (3), but successive treatment in reverse order was somewhat less effective. No combinational treatment upregulated granulocytic lactate dehydrogenase, Gr-1, or chloroacetate esterase to as great an extent as was obtained with granulocyte colony-stimulating factor alone, indicating that granulocytic differentiation is attenuated by addition of 1,25-dihydroxyvitamin D (3). Therefore, in contrast to our previous data, the present findings suggest that granulocyte colony-stimulating factor synergistically augments 1,25-dihydroxyvitamin D (3)-induced monocytic differentiation in our murine bone-marrow cell cultures. Considering previously published data, we also suggest that these synergistic effects may be mainly due to the combination of two distinct effects such as the primary proliferative effects of granulocyte colony-stimulating factor on multipotent stem cells and the subsequent differentiative effects of 1,25-dihydroxyvitamin D (3) on proliferating cells.     

Growth of mouse and human bone marrow in diffusion chambers in mice. Development of myeloid and erythroid colonies and proliferation of myeloid stem cells in cyclophosphamide- and erythropoietin-treated mice.

Both murine and human bone marrow cells were cultured in plasma clots which were formed inside diffusion chambers implanted into cyclophosphamide- and saline-treated mice. After an initial fall, the number of mouse bone marrow cells and numbers of mouse myeloid stem cells (CFU-C) and agar cluster-forming units rose faster in the cyclophosphamide-treated animals. These hosts also favored formation of myeloid (CFU-D-G) and erythroid (CFR-D-E) colonies and myeloid higher than those of CFU-C from the same marrow population. These observations suggest the existence of humoral factors stimulating granulocyte progenitor cell replication and differentiation. At its best the increment of CFU-D-E number was equivalent to that caused by a single 0.1 unit erythropoietin dose. Culture of normal human marrow cells resulted in colonies in the plasma clot containing only granulocytes and macrophages. Cyclophosphamide-treated host animals were essential for human CFU-D-G development. Plating efficiency for human marrow myeloid colonies was better in the conventional in vitro agar cultures than in diffusion chambers.     

Control of normal differentiation of myeloid leukemic cells. XII. Isolation of normal myeloid colony-forming cells from bone marrow and the sequence of differentiation to mature granulocytes in normal and D+ myeloid leukemic cells

An enriched population of early myeloid cells has been obtained from normal mouse bone marrow by injection of mice with sodium caseinate and the removal of cells with C3 (EAC) rosettes by Ficoll-Hypaque density centrifugation. This enriched population had no EAC or Fc (EA) rosettes and contained 87% early myeloid cells stained for myeloperoxidase and/or AS-D-chloroacetate esterase, 7% cells in later stages (ring forms) of myeloid differentiation and 6% unstained cells, 2% of which were small lymphocytes. After seeding in agar with the macrophage and granulocyte inducer MGI, the enriched population showed a cloning efficiency of 14% when removed from the animal and of 24% after one day in mass culture. Both the enriched and the unfractionated bone marrow cells gave the same proportion of macrophage and granulocyte colonies. The normal early myeloid cells were induced to differentiate by MGI in mass culture in liquid medium to mature granulocytes and macrophages. The sequence of granulocyte differentiation was the formation of EA and EAC rosettes followed by the synthesis and secretion of lysozyme and morphological differentiation to mature cells. D⁺ myeloid leukemic cells with no EA or EAC rosettes had a similar morphology to normal early myeloid cells and showed the same sequence of differentiation. The induction of EA and EAC rosettes occurred at the same time in both the normal and D⁺ leukemic cells, but lysozyme synthesis and the formation of mature granulocytes was induced later in the leukemic than in the normal cells. The results indicate that selection for non-rosette-forming normal early myeloid cells also selected for myeloid colony forming cells, that these normal early myeloid cells can form colonies with differentiation to macrophages and granulocytes, that normal and D⁺ myeloid leukemic cells have a similar sequence of differentiation and that the normal cells had a greater sensitivity for the formation of mature cells by MGI.     

An increase in intracellular pH is a general response of promonocytic cells to differentiating agents

Intracellular pH (pHi) was measured in HL60 and U937 cells before and after differentiation into monocyte-macrophage like cells. 12-O-Tetradecanoyl phorbol-13-acetate (PMA), butyrate, interferon, retinoic acid and 1,25-dihydroxyvitamin D3 all increased pHi. The increases elicited were rapid with PMA, much slower with retinoic acid and interferon and still slower with 1,25-dihydroxyvitamin D3. Increases in pHi are due to an activation of the Na+/H+ exchange system. High pHi values are unlikely to serve as an early intracellular signal for initiating monocytic differentiation.     

Identification of a common signal associated with cellular proliferation stimulated by four haemopoietic growth factors in a highly enriched population of granulocyte/macrophage colony-forming cells.

We have prepared a population of bone marrow cells that is highly enriched in neutrophil/macrophage progenitor cells (GM-CFC). Four distinct haemopoietic growth factors can stimulate the formation of mature cells from this population, although the proportions of neutrophils and/or macrophages produced varied depending on the growth factor employed: interleukin 3 (IL-3) and granulocyte/macrophage colony-stimulating factor (GM-CSF) stimulated the formation of colonies containing both neutrophils and macrophages; macrophage colony-stimulating factor (M-CSF) produced predominantly macrophage colonies; and granulocyte colony-stimulating factor (G-CSF) promoted neutrophil colony formation. Combinations of these four growth factors did not lead to any additive or synergistic effect on the number of colonies produced in clonal soft agar assays, indicating the presence of a common set of cells responsive to all four haemopoietic growth factors. These enriched progenitor cells therefore represent an ideal population to study myeloid growth-factor-stimulated survival, proliferation and development. Using this population we have examined the molecular signalling mechanisms associated with progenitor cell proliferation. We have shown that modulation of cyclic AMP levels has no apparent role in GM-CFC proliferation, whereas phorbol esters and/or Ca2+ ionophore can stimulate DNA synthesis, indicating a possible role for protein kinase C activation and increased cytosolic Ca2+ levels in the proliferation of these cells. The lack of ability of all four myeloid growth factors to mobilize intracellular Ca2+ infers that these effects are not achieved via inositol lipid hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

1,25-Dihydroxyvitamin D3 and the regulation of the differentiation and function of macrophages and granulocytes

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) exerts a differential inhibitory effect on the formation of granulocyte, granulocyte/macrophage, and macrophage colonies grown from mouse bone marrow precursor cells; 50% inhibition was attained at 1.1, 2.3, and 23 nM 1,25(OH)2D3, respectively. The inhibition of colony formation, as well as phagocyte proliferation in liquid cultures, requires the presence of 1,25(OH)2D3 in the early stages of culture (up to 72 h after culture initiation). 1,25(OH)2D3 induces a dose- and time-dependent augmentation of the phagocytic capability of mononuclear phagocytes (up to 100%) towards both heat-killed yeast cells and IgG-coated sheep red blood cells. The augmentation of the phagocytic capability of the mononuclear phagocytes depends critically on when 1,25(OH)2D3 is added. It is effective when added up to 72 h after culture initiation, while at later stages (greater than or equal to 96 h) the cells are no longer induced to express enhanced phagocytic capability. We suggest that these phenomena may be relevant to hemopoietic processes.     

1 alpha, 25-Dihydroxyvitamin D3 augments clonal growth of macrophages from rat bone marrow progenitor cells and modulates their function

1 alpha, 25-Dihydroxyvitamin D3 (1,25(OH)2D3) was shown to enhance (approximately 2 fold) the colony-stimulating factor-dependent clonal growth of macrophage colonies and clusters from rat bone marrow progenitor cells. The proliferative capacity of macrophage progenitors in liquid cultures was likewise augmented (2-3 fold). Mononuclear phagocytes (macrophages, for simplicity) developing in the presence of 1,25(OH)2D3 showed a reduced capacity of migration. 1,25(OH)2D3 administered at bone marrow culture initiation led to augmentation of the phagocytic capability of macrophages in four-day cultures and to its suppression in macrophages in seven-day cultures. The observed patterns of modulation of differentiation and function by 1,25(OH)2D3 differ from the patterns we found for mouse bone marrow cells. The results suggest that the differential response to hormones observed in different species may include responses to 1,25(OH)2D3.