Human platelet alpha granules contain a nonspecific inhibitor of megakaryocyte colony formation: its relationship to type beta transforming growth factor (TGF-beta)

Whole blood serum (WBS) and platelet-poor plasma-derived serum (PDS) from the same normal subject were compared for their abilities to support human megakaryocyte (MK) colony formation. In all cases, PDS promoted the growth of a higher number (20-50%) of MK colonies than did WBS. Increasing amounts of WBS decreased the number of colonies, whereas increasing concentration of PDS had no marked effects. Crude platelet extracts or platelet secretory products from thrombin-activated platelets also elicited an inhibition of MK colony formation in a dose-dependent manner. A complete inhibition was found for a dose equivalent to 1.10(9) platelets/ml and a 50% inhibition in a range of 1.10(7)-1.10(8) platelets/ml. These platelet products were also inhibitory for erythroid progenitor growth. Platelets from two patients with gray platelet syndrome elicited only a minor inhibition of MK growth, suggesting that the platelet alpha granule is the origin of this inhibition. When platelet extracts were acid-treated, the biological activity of the inhibitor on CFU-MK and CFU-E growth was 20-50-fold higher. In addition, a potent stimulatory activity on the growth of day 7 CFU-GM was observed. The enhancement of biological activities by acid treatment suggests that type beta transforming growth factor (TGF-beta) could be involved in this platelet inhibitory activity. The homogeneous native TGF-beta (from 1 pg to 1 ng/ml) produced the same effects previously induced by platelet products. It totally inhibited CFU-MK growth (at a 500 pg/ml), it inhibited CFU-E growth, and it stimulated growth of day 7 CFU-GM in the presence of a colony-stimulating factor. The inhibition of CFU-MK growth was also observed on purified progenitors. In conclusion, these results suggest that TGF-beta may be implicated in negative autocrine regulation of megakaryopoiesis. However, since this molecule has ubiquitous biological activities, its physiologic relevance as a normal regulator of megakaryopoiesis requires further investigation.     

Activated charcoal diminishes the lot difference of fetal bovine sera in erythroid colony formation of human bone marrow cells

Using normal bone marrow as target cells, we assayed the colony-forming efficiency of early and late erythroid progenitor cells and granulocyte-macrophage progenitor cells using several different lots of fetal bovine serum (FBS). There was a marked difference in the ability of these sera to support colony formation, particularly in erythroid colony assays. When adsorbed by activated charcoal, all these sera supported erythroid colony formation more efficiently than before adsorption. There was no significant effect of charcoal adsorption of FBS on granulocyte-macrophage colony formation. Gel-filtration study showed that charcoal adsorption diminished low-molecular-weight fractions by less than 5000 Da. The inhibitory activity of this fraction was heat labile and Pronase sensitive. Concentrated samples obtained from these fractions inhibited erythroid colony formation in a dose-dependent manner. These results suggest that low-molecular-weight inhibitors that are relatively specific to erythropoiesis play a critical role in the lot differences of FBS for erythroid colony formation.     

Recombinant human tumor necrosis factors alpha and beta stimulate fibroblasts to produce hemopoietic growth factors in vitro

The influences of TNF alpha and TNF beta were evaluated for their stimulatory and inhibitory effects on in vitro colony formation by human bone marrow granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells. Both TNF alpha and TNF beta induced fibroblasts to produce stimulators of CFU-GM, BFU-E, and CFU-GEMM in a dose-dependent fashion. Similar results were seen when equivalent concentrations of TNF alpha and TNF beta were used. Prior incubation of the TNF alpha and TNF beta with their respective antibodies inactivated the ability of the TNF preparations to induce the release of granulocyte-macrophage, erythroid, and multipotential colony-stimulating activity from fibroblasts. In addition, incubation of the TNF-induced fibroblast supernatant with antibody before colony assay resulted in enhanced colony formation, suggesting that the TNF carried over into the colony assay suppressed colony formation. Additional proof of this suppression by TNF was evident when TNF was added directly to the CFU-GM, BFU-E, and CFU-GEMM colony assays. IL-1 does not appear to function as an intermediary in growth factor production by fibroblasts stimulated with TNF because antibody to IL-1 displayed no effect. Furthermore, assay of TNF-induced fibroblast supernatant was negative for IL-1. These results suggest that TNF alpha and TNF beta exert both a positive and negative influence on in vitro hemopoietic colony formation.     

The suppressive influences of human tumor necrosis factors on bone marrow hematopoietic progenitor cells from normal donors and patients with leukemia: synergism of tumor necrosis factor and interferon-gamma

The influences of human tumor necrosis factor (TNF) (LuKII), recombinant human TNF-alpha, natural human interferon-gamma (HuIFN-gamma), recombinant HuIFN-gamma, and natural HuIFN-alpha were evaluated alone or in combination for their effects in vitro on colony formation by human bone marrow granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells incubated at 5% CO2 in lowered (5%) O2 tension. TNF (LuKII) and recombinant TNF-alpha caused a similar dose-dependent inhibition of colony formation from CFU-GM, BFU-E, and CFU-GEMM. Day 7 CFU-GM colonies were more sensitive than both day 14 CFU-GM colonies and day 7 CFU-GM clusters to inhibition by TNF. BFU-E colonies and CFU-GEMM colonies were least sensitive to inhibition with TNF. The suppressive effects of TNF (LuKII) and recombinant TNF-alpha were inactivated respectively with hetero-anti-human TNF (LuKII) and monoclonal anti-recombinant human TNF-alpha. The hetero-anti-TNF (LuKII) did not inactivate the suppressive effects of TNF-alpha and the monoclonal anti-recombinant TNF-alpha did not inactivate TNF (LuKII). The suppressive effects of TNF did not appear to be mediated via endogenous T lymphocytes and/or monocytes in the bone marrow preparation, and a pulse exposure of marrow cells with TNF for 60 min resulted in maximal or near maximal inhibition when compared with cells left with TNF for the full culture incubation period. A degree of species specificity was noted in that human TNF were more active against human marrow CFU-GM colonies than against mouse marrow CFU-GM colonies. Samples of bone marrow from patients with non-remission myeloid leukemia were set up in the CFU-GM assay and formed the characteristic abnormal growth pattern of large numbers of small sized clusters. These cluster-forming cells were more sensitive to inhibition by TNF than were the CFU-GM colonies and clusters grown from the bone marrow of normal donors. The sensitivity to TNF of colony formation by CFU-GM of patients with acute myelogenous leukemia in partial or complete remission was comparable with that of normal donors. When combinations of TNF and HuIFN were evaluated together, it was noted that TNF (LuKII) or recombinant TNF synergized with natural or recombinant HuIFN-gamma, but not with HuIFN-alpha, to suppress colony formation of CFU-GM, BFU-E, and CFU-GEMM from bone marrow of normal donors at concentrations that had no suppressive effects when molecules were used alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effect of vitamin D3 metabolites on normal and leukemic bone marrow cells in vitro

We studied the effects of 1,25-dihydroxyvitamin D3 and other metabolites of vitamin D3 on the maturation in liquid culture and on colony formation in semisolid media of marrow and buffy coat cells from patients with myeloid leukemias and from normal individuals. After incubation with 1,25-dihydroxy-vitamin D3, a proportion of both normal and leukemic myeloid cells resembled cells of the monocyte-macrophage lineage; these cells expressed alpha-naphthylacetate esterase and were able to phagocytize and kill candida organisms. When granulocyte-macrophage progenitor cells (CFU-GM) were incubated with 1,25-dihydroxyvitamin D3, the number of monocyte-macrophage colonies was increased and the number of granulocyte colonies was reduced; megakaryocyte colony formation (CFU-Mk) was inhibited substantially; and there was no effect on erythroid (BFU-E) or multilineage (CFU-GEMM) progenitor cell colony formation. We propose that 1,25-dihydroxyvitamin D3 may induce cells that are normally committed to differentiate along the granulocytic pathways to differentiate instead along the monocyte-macrophage pathway. If these in vitro observations reflect the in vivo activity of 1,25-dihydroxyvitamin D3, it may be involved in the modulation of collagen deposits in the bone marrow.     

Sensitivity of Ph 1 + CFU-GM to human recombinant interferon alpha and gamma alone and in combination

The in vitro effect of human recombinant interferon alpha (IFN) alone and in combination were studied on granulomonocytic colony forming units (CFU-GM) from the peripheral blood of 10 Ph 1+ chronic myeloid leukemia (CML) patients and from the marrow of 5 normal or non-leukemic subjects. alpha- and gamma-IFN alone determined a slight inhibition on colony growth with a preferential effect on "pure" macrophagic colonies. At maximum concentration (10(4) U/ml) leukemic colony inhibition was 46 +/- 34% for alpha IFN and 43 +/- 19% for gamma IFN. Culture growth with alpha + gamma IFN in combination were significantly inhibited (up to 96 +/- 4%) with a concentration-related effect. Similar results were obtained with normal CFU-GM. The synergism that was found in vitro is probably relevant for the in vivo therapeutic effects of these compounds in CML and suggest that the combination is worth testing in vivo.     

Growth factors in bone marrow transplant recipients

Normal human plasma samples usually support the growth of hemopoietic progenitors in the presence of exogenous growth factors. In contrast, plasma samples collected serially from bone marrow transplant recipients after administration of their preparative regimen demonstrate activities that routinely stimulate the growth of granulocyte-macrophage progenitors (CFU-GM) and precursors for megakaryocytes (CFU-Meg). The peak activities are usually observed between day 7 and day 21. Occasionally growth promoting activities for erythroid bursts are also observed. Activities for the growth of multilineage colonies are only rarely seen. These growth promoting activities for CFU-GM and CFU-Meg usually return to pretransplant values by day 30 in patients with prompt engraftment. Patients with delayed engraftment sustain high levels of growth promoting activities for long periods of time.     

Predominant suppression of neutrophil colony growth by recombinant human tumor necrosis factor

To investigate the suppressive effect of recombinant human tumor necrosis factor (rH-TNF) on colony growth of human granulocyte-macrophage progenitor cells (CFU-GM), cytochemical examinations of CFU-GM colonies were performed by a triple staining method. Each colony was classified into five subtypes, and the effects of rH-TNF on each subtype were analyzed. Neutrophil colony growth was inhibited by rH-TNF in a dose-dependent manner, and it was almost completely suppressed at 100 U/ml. In contrast, no significant suppressive effect of rH-TNF was found on the growth of monocyte-macrophage and eosinophil colonies at 100 U/ml or less. When recombinant human granulocyte colony-stimulating factor which almost exclusively stimulates neutrophil colony formation was used as a source of colony-stimulating activity, the total colony growth was almost completely suppressed by 100 U/ml of rH-TNF. These results indicate predominant inhibition of neutrophil colony growth by rH-TNF.     

Transforming growth factor beta: possible roles in the regulation of normal and leukemic hematopoietic cell growth

We have recently demonstrated that transforming growth factor (TGF)-beta 1 and TGF-beta 2 are potent inhibitors of the growth and differentiation of murine and human hematopoietic cells. The proliferation of primary unfractionated murine bone marrow by interleukin-3 (IL-3) and human bone marrow by IL-3 or granulocyte/macrophage colony-stimulating factor (GM-CSF) was inhibited by TGF-beta 1 and TGF-beta 2, while the proliferation of murine bone marrow by GM-CSF or murine and human marrow with G-CSF was not inhibited. Mouse and human hematopoietic colony formation was differentially affected by TGF-beta 1. In particular, CFU-GM, CFU-GEMM, BFU-E, and HPP-CFC, the most immature colonies, were inhibited by TGF-beta 1, whereas the more differentiated unipotent CFU-G, CFU-M, and CFU-E were not affected. TGF-beta 1 inhibited IL-3-induced growth of murine leukemic cell lines within 24 h, after which the cells were still viable. Subsequent removal of the TGF-beta 1 results in the resumption of normal growth. TGF-beta 1 inhibited the growth of factor-dependent NFS-60 cells in a dose-dependent manner in response to IL-3, GM-CSF, G-CSF, CSF-1, IL-4, or IL-6. TGF-beta 1 inhibited the growth of a variety of murine and human myeloid leukemias, while erythroid and macrophage leukemias were insensitive. Lymphoid leukemias, whose normal cellular counterparts were markedly inhibited by TGF-beta, were also resistant to TGF-beta 1 inhibition. These leukemic cells have no detectable TGF-beta 1 receptors on their cell surface. Last, TGF-beta 1 directly inhibited the growth of isolated Thy-1-positive progenitor cells. Thus, TGF-beta may be an important modulator of normal and leukemic hematopoietic cell growth.     

Suppression of maturation of megakaryocyte colony forming unit in vitro by a platelet-released glycoprotein

The suppressive role of platelets on the growth of human marrow megakaryocyte colony forming units (CFU-M) in vitro was investigated by the use of a plasma clot assay. An inverse correlation was established between the number of megakaryocytic colonies grown and the platelet concentration of the plasma or the resultant serum used in the culture system. The suppressive effect of platelets on megakaryocyte colony formation reached a plateau at normal human blood platelet concentration and was specific for CFU-M growth, since marrow cell erythroid burst formation (BFU-E) and granulocytic-monocytic colony formation (CFU-GM) remained unaffected. The inhibitory activity was detectable in the supernatants of platelet suspensions aggregated by thrombin or ADP, and the inhibitory activity released from ADP-stimulated platelets was blocked by pretreatment of platelets with monoclonal antibody HuPl-m1. Partial purification of this activity was achieved by diethylaminoethyl (DEAE)-ion exchange and phytohemagglutinin (PHA)-E agarose affinity chromatography. This inhibitor is a glycoprotein with a molecular weight of 12-17K daltons. This platelet released glycoprotein does not affect the early proliferative phase of CFU-M in vitro but acts on a day 6-8 CFU-M-derived cell by adversely affecting its maturation into recognizable megakaryocytes. These findings demonstrate that a glycoprotein released from platelets suppresses the maturation of CFU-M into megakaryocytes.     

Effects of okadaic acid on mouse hemopoietic cells

Effects of okadaic acid, a potent non-12-O-tetradecanoyl-phorbol-13-acetate(TPA)-type tumor promoter, on mouse hemopoietic cells were investigated. Okadaic acid stimulated mouse bone marrow cells to form granulocyte-macrophage colony-forming unit (CFU-GM) colonies without added colony stimulating factors(CSFs). At the concentration of 1.82 x 10(-8) M, colony formation of 77 +/- 14 colonies/1 x 10(5) bone marrow cells was observed. Observations on the effects of other cells on the CSF induction suggested that okadaic acid primarily stimulated the functions of macrophages, and the CSF production from macrophages might be attributed to the CFU-GM colony formation. On the other hand, the erythroid colony-forming unit(CFU-E) colony formation stimulated by     

Deficiency of pluripotent hemopoietic progenitor cells in myelodysplastic syndromes

Pluripotent (CFU-MIX), erythroid (BFU-E) and granulocyte/macrophage (CFU-GM) progenitor cells were examined in bone marrow (BM) from 23 patients with myelodysplastic syndromes (MDS). Patients were grouped according to the FAB classification: Refractory anemia (RA), n = 3; RA with ring sideroblasts (RARS), n = 3; RA with excess of blasts (RAEB), n = 8; RA with excess of blasts in transformation (RAEBt), n = 7; chronic myelomonocytic leukemia (CMML), n = 2. In FAB groups RA, RARS, RAEB and RAEBt CFU-GM concentrations were normal or decreased but both CMML-patients had increased CFU-GM values. Abnormal cluster growth was observed in 9 of 23 MDS-patients. BFU-E colony formation was subnormal in all cases. Mixed-colony assay values were at the lower limit of controls in one patient and decreased in the remaining 22 MDS-patients. A similar growth pattern of hemopoietic progenitor cells was observed in 19 patients with acute nonlymphocytic leukemia (ANLL), who were studied for comparison. These data suggest a quantitative or qualitative/functional defect of the pluripotent progenitor cell compartment as the major cause for the cytopenia in MDS-patients.     

The anemia of thermal injury: mechanism of inhibition of erythropoiesis

The anemia of thermal injury is a multifactorial process and includes hemorrhage and hemolysis. Much evidence suggests that a reduced rate of erythropoiesis contributes to this anemia. Prior studies show that this anemia is temporally related to the appearance in burn patients sera of a substance(s) capable of inhibiting erythropoiesis in vitro. Four experiments were done to elucidate the mechanism of action of this inhibitor. In all experiments sera from burn patients previously shown to be inhibitory to erythropoiesis in vitro were studied. In the first, inhibitory sera were exposed to erythropoietin solutions without loss of erythropoietic activity. Second, mouse marrow cells were preincubated with serum without loss of their ability to form erythroid colonies. Third, the inhibitory effect could not be overcome with increasing amounts of erythropoietin. Finally, erythroid colony formation was effected only if the inhibitory serum was present during the first 8 to 12 hr of culture. The data suggest that the erythropoietic inhibitor in these sera acts directly on erythroid stem cells in vitro and not by inactivating or interference with erythropoietin.     

In vitro culture studies of granulocyte/macrophage and erythroid progenitor cells in lymphoproliferative disorders

Granulocyte/macrophage progenitor cells (CFU-GM) and erythroid progenitor cells (BFU-E) have been assayed in peripheral blood (PB) and/or bone marrow (BM) from 12 patients with acute lymphocytic leukemia (ALL), 16 patients with chronic lymphocytic leukemia (CLL) and 31 patients with various forms of non-Hodgkin lymphoma (NHL) without BM involvement. Progenitor cell growth in PB and BM from the NHL patients did not differ statistically from controls (p greater than 0.1). CFU-GM and BFU-E per ml PB were markedly increased in ALL and CLL patients (p less than 0.001) while CFU-GM and BFU-E per plated BM cells from these patients were severely depressed (p less than 0.001). Lymphoblasts from one ALL patient failed to inhibit CFU-GM and BFU-E-derived colony growth from control PB mononuclear cells. The high levels of circulating progenitor cells in ALL and CLL patients clearly distinguish them from other cytopenic hematological malignancies, in which decreased progenitor cell levels have been demonstrated previously (acute myeloid leukemia, hairy cell leukemia). The cause of this finding and its pathophysiological implication still remains to be established.     

Effects of purified recombinant human interleukin-3 on the growth of human hematopoietic progenitor cells in "long-term" bone marrow culture

Recombinant human interleukin-3 (rhuIL-3) was assessed for its effects on the growth of normal human hematopoietic bone marrow nucleated cells, and on granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells in a liquid culture system which allows for the prolonged growth of these cells in vitro. RhuIL-3, at concentrations of 100 and 500 units/mL, significantly enhanced the numbers of nucleated cells, as well as the numbers of supernatant and adherent CFU-GM and BFU-E growing in tissue culture flasks or dishes over a period of 4 to 6 weeks. The results demonstrated the rhuIL-3 has a stimulating effect on the growth of human marrow cells in prolonged culture. This information is consistent with the effects of rhuIL-3 in short-term marrow colony assays in vitro and with the in vivo actions of recombinant murine IL-3 in mice, and may be of relevance to clinical trials that will be assessing the hematopoietic effects of rhuIL-3 in humans.     

Influence of IL-1 alpha and -1 beta on the survival of human bone marrow cells responding to hematopoietic colony-stimulating factors

Purified recombinant human (rhu) IL-1 alpha and IL-1 beta were evaluated for their effects on the proliferation and survival of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E) progenitor cells from normal human bone marrow (BM). Using nonadherent low density T lymphocyte depleted (NALT-) BM cells cultured in the presence or absence of IL-1, CSF-deprivation studies demonstrated that IL-1 alpha or IL-1 beta by itself did not enhance the proliferation of CFU-GM or BFU-E. They did, however, promote the survival of progenitors responding to the delayed addition of media conditioned by the 5637 cell line (5637 conditioned medium), rhu GM-CSF and erythropoietin. The survival promoting effects of IL-1 alpha on CFU-GM and BFU-E were neutralized by anti-IL-1 alpha mAb added to the cultures. The survival promoting effect of IL-1 alpha did not appear to be mediated by CSF, because neither CSF nor erythroid burst promoting activity were detectable in cultures in which NALT- cells were incubated with rhuIL-1 alpha. In addition, suboptimal concentrations of rhu macrophage CSF (CSF-1), G-CSF, GM-CSF, and IL-3, which were just below the levels that would stimulate colony formation, did not enhance progenitor cell survival. Survival of CFU-GM and BFU-E in low density (LD) bone marrow cells did not decrease as drastically as that in NALT- BM cells, and exogenously added IL-1 did not enhance progenitor cell survival of CFU-GM and BFU-E in LD BM cells. However, addition of anti-IL-1 beta decreased survival of CFU-GM and BFU-E in LD BM cells. These results implicate IL-1 in the prolonged survival of human CFU-GM and BFU-E.     

Expression of hematopoietic inhibitory factors in mouse fibroblasts,macrophages and endothelial cells

Pure bone marrow fibroblasts, macrophages and endothelial cells were cultured in Iscove-modified Dulbecco's medium. RT-PCR was used to determine the expression of inhibitory cytokine mRNAs in these cell types. Serum-free conditioned medium was collected from each cell type and ultrafiltration was performed with a centriprep 10. The retentate contained substances whose molecular weights were >10 kD, whilst the filtrate contained substances with molecular weights <10 kD. The effect of conditioned media and their components on colony forming unit-granulocyte-macrophage (CFU-GM) were investigated. The results showed: (1) six cytokines, MIP-1alpha, MIP-2, TGF-beta, TNF-alpha, IFN-gamma and Tbeta(4), inhibited the growth of CFU-GM when murine WEHI-3 conditioned medium was added to the culture system as a source of colony stimulation. (2) The original endothelial cell conditioned medium (E-CM) did not affect the production of CFU-GM, but the >10 kD component of E-CM increased its production, and the <10 kD component decreased it. Both fibroblast conditioned medium (F-CM) and the >10 kD component of F-CM stimulated proliferation of CFU-GM, but the <10 kD component suppressed it. All three components of macrophage conditioned medium (M-CM) inhibited the growth of CFU-GM. (3) Expression of four of the mRNAs, namely MIP-2, TNF-alpha, INF-gamma and Tbeta(4), was seen in all three types of stromal cells, while TGF-beta mRNA was only seen in endothelial cells and macrophages, and MIP-1alpha mRNA in endothelial cells and fibroblasts. The inhibitors TGF-beta, MIP-1alpha, and Tbeta(4)have an inhibitory effect on the growth of CFU-GM, but TNF-alpha, INF-gamma and MIP-2 do not.     

HLA-DR restriction in suppression of hematopoiesis by T cells from allogeneic bone marrow transplants

Three allogeneic bone marrow transplantation patients who exhibited a suppressive subset of T cells for in vitro hematopoiesis have been investigated to determine whether this T cell suppressive effect was genetically restricted. In the three cases, T cells separated by sheep red cell rosetting inhibited blood colony-forming units granulocyte-monocyte (CFU-GM) and burst-forming unit erythroid (BFU-E) growth from the patients and from the bone marrow donors who were HLA identical, but not from randomly chosen unrelated subjects. In one case, cocultures were performed between the patient T cells and the T-depleted cells from eight siblings and from the mother. A marked inhibition (30 to 60%) of CFU-GM and BFU-E growth was found in the relatives who shared a haplo-identical HLA-DR 5. The same degree of suppression was found with respect to whether the siblings were homozygous or heterozygous for the HLA-DR 5 antigen, and whether or not they shared common class I antigens. This inhibition was totally abolished when a monoclonal antibody against HLA-DR was added, whereas a monoclonal antibody against class I histocompatibility antigen had no effect. To additionally demonstrate that this inhibition was mediated by a single HLA-DR haplotype, T cells from the patient were co-cultured with cells from three normal unrelated individuals, one with a phenotypically identical DR and two with only one haploidentical DR. Inhibition was similarly found in the subject exhibiting complete DR identity, and the subject with only the DR 5 haploidentical phenotype. These results demonstrate that a unique subset of T cells present in allogeneic bone marrow transplants specifically suppress differentiation of hemopoietic progenitors that bear one phenotypically haplo-identical HLA-DR antigen.