Protection of 3'-azido-3'-deoxythymidine induced toxicity to murine hematopoietic progenitors (CFU-GM, BFU-E and CFU-MEG) with interleukin-1

3'-Azido-3'-deoxythymidine (AZT) has attained wide clinical utility in the treatment of acquired immunodeficiency syndrome (AIDS). Unfortunately, associated with AZT use, is the development of severe hematopoietic toxicity as manifested by anemia, neutropenia and overall bone marrow suppression. Interleukin-1 (IL-1), a cytokine, primarily produced by activated macrophages, has been involved in the control of hematopoiesis by acting synergistically with other hematopoietic growth factors, and has been demonstrated to be an effective agent in reducing the myelosuppression associated with the therapy for malignant disease. We report here the ability of recombinant human IL-1 alpha to protect normal murine hematopoietic progenitors (CFU-GM, BFU-E, and CFU-Meg) from the toxic effects of AZT. Following the determination of the LD50 dose for each progenitor, IL-1 was added in co-culture studies (10-1000 units; 0.001-1.0 micrograms/ml protein) with adherent cell depleted marrow. Marrow progenitors expressed differences in AZT sensitivity, e.g., BFU-E, LD50 5 x 10(-9)M; CFU-Meg, LD50 10(-7) M; CFU-GM, 5 x 10(-5) M respectively. IL-1 inhibited AZT induced toxicity. The maximum IL-1 dose effect was observed for CFU-GM and CFU-Meg at 300 units, 0.3 micrograms protein; however BFU-E required a dose of 600 units, 0.6 micrograms/ml protein to reverse the effects of AZT. These results demonstrate marrow progenitors respond differently to AZT and identifies the potential efficacy of IL-1 to minimize the hematopoietic toxicity associated with AZT treatment.     

Hematopoiesis in the rat: quantitation of hematopoietic progenitors and the response to iron deficiency anemia

To determine the quantitative effects of iron deficiency on erythropoiesis and to assess the response of erythroid progenitors to sustained anemia, we developed quantitative assays for various hematopoietic progenitors in the adult, Sprague-Dawley rat including erythroid colony- and burst-forming cells (CFU-E and BFU-E), granulocyte/macrophage colony-forming cells (CFU-GM), and megakaryocytic colony-forming cells (CFU-Meg). CFU-E were cultured in methylcellulose and grew best in the presence of fetal calf serum. CFU-GM, BFU-E, and CFU-Meg grew better in normal rat plasma and required the presence of pokeweed mitogen-stimulated rat spleen cell conditioned medium. The numbers of progenitors and nucleated erythroblasts in total marrow were estimated by the ratios of radioactivity in the humerus to the total skeleton as determined by radioiron dilution. The numbers of progenitors and erythroblasts in the spleen were measured by simple dilution. Sustained anemia was brought about through chronic iron deficiency. The response to iron deficiency anemia (IDA) was monitored by the numbers of the various progenitors and their cell cycle characteristics as measured by the tritiated thymidine suicide technique. With IDA, the number of CFU-F in the body (marrow plus spleen) was increased to 3.5 times control, whereas the numbers of BFU-E and CFU-GM were unchanged. There was no difference in the percentage of CFU-E, BFU-E, and CFU-GM in DNA synthesis (68%, 19.4%, and 18.8%, respectively). With iron therapy of IDA, CFU-E numbers in marrow began to decrease by day 1 and fell in a manner reciprocal to changes in the hematocrit. Marrow and spleen erythroblasts, 1.7 times control in IDA, increased further to 3.9 times control by the fourth day after iron administration. There was no change in BFU-E or CFU-GM numbers in response to iron repletion, although the fraction of progenitors increased in the spleen. Thus, IDA does not limit the increase in CFU-E seen with anemia, but does restrict erythroid maturation. Furthermore, the increase in CFU-E and the state of chronic anemia occur without detectable changes in the number of cell cycle state of the more primitive BFU-E.     

Susceptibility to merocyanine 540-mediated photosensitization: a differentiation marker on murine hematopoietic progenitor cells

Merocyanine 540 (MC 540) is an impermeant fluorescent dye that binds preferentially to fluidlike domains of the cell membrane. Photoexcitation of membrane-bound dye causes a breakdown of the normal permeability properties of the membrane and, eventually, cell death. We have used in vitro and in vivo clonal assays to determine the relative sensitivities of different classes of normal murine hematopoietic progenitor cells to MC 540-mediated photosensitization. Late erythroid progenitors (CFU-E) were the most sensitive cells, followed in order of decreasing sensitivity by early erythroid progenitors (BFU-E), megakaryocyte progenitors (CFU-Meg), day 7-spleen colony forming cells (day 7-CFU-S), granulocyte/macrophage progenitors (CFU-GM), and day 11-spleen colony forming cells (day 11-CFU-S). Bipotent progenitors of the granulocyte/macrophage lineage were more sensitive than unipotent macrophage progenitors but less sensitive than unipotent granulocyte progenitors. Progenitors giving rise to large granulocyte/macrophage colonies were more sensitive than progenitors giving rise to small colonies ("clusters"). We conclude that sensitivity to MC 540-mediated photosensitization is develop-mentally regulated and that differences occur even between the most closely related classes of progenitor cells. Our findings indicate the usefulness of MC 540 as a plasma membrane probe. They also support the contention that early and late-appearing spleen colonies are the progeny of two distinct classes of progenitor cells.     

小鼠骨髓内皮细胞分泌的细胞因子对造血干/祖细胞增殖的影响

应用小鼠骨髓内皮细胞株细胞传代培养,收集无血清条件培养液（ｍＢＭＥＣ－ＣＭ）,经超滤分成大于１０ｋＤ和小于１０ｋＤ两组分,分别观察两组分的ｍＢＭＥＣ－ＣＭ对小鼠骨髓造血干／祖细胞ＣＦＵ－ＧＭ,ＨＰＰ－ＣＦＣ,ＣＦＵ－Ｅ,ＢＦＵ－Ｅ和ＣＦＵ－Ｍｅｇ的影响。结果表明：含分子量大于１０ｋＤ物质的ｍＢＭＥＣ－ＣＭ的保留液能明显刺激ＣＦＵ－ＧＭ,ＨＰＰ－ＣＦＣ,ＣＦＵ－Ｅ,ＢＦＵ－Ｅ和ＣＦＵ－Ｍｅｇ生长;     

Suppression of murine hematopoiesis in vivo after chronic administration of zidovudine: evidence that zidovudine-induced anemia is the result of decreased bone marrow-derived, erythropoietin-responsive progenitor cells.

We studied the long-term effect of continued zidovudine exposure in mice on hematopoiesis, as determined by peripheral blood indices, assays of erythroid (colony-forming unit-erythroid [CFU-E] and burst-forming unit-erythroid [BFU-E]), myeloid (CFU-granulocyte-macrophage [GM]), megakaryocyte (CFU-Meg), and plasma titers of erythropoietin, granulocyte-macrophage colony-stimulating factor, megakaryocyte colony-stimulating factor, and tumor necrosis factor-alpha. Dose-escalation of zidovudine (0.1, 1.0, and 2.5 mg/ml) induced a dose-dependent decrease in hematocrit, white blood cells, and platelets. High-dose drug, i.e., greater than 1.0 mg/ml, reduced marrow CFU-E; splenic CFU-E was increased after 1 week, then declined. BFU-E was increased at Weeks 1 and 2, then declined to control levels. Splenic BFU-E rose during the examination period that was dose-dependent. Femoral CFU-GM was cyclic, i.e., low-dose drug, 0.1 mg/ml, was increased gradually, the declined; higher doses of 1.0 and 2.5 mg/ml were lower until Week 5, then were above controls. Splenic CFU-GM was increased initially at Week 2 (1.0 mg/ml), then declined; the higher dose (2.5 mg/ml) increased initially, then declined below controls (Week 6). Femoral CFU-Meg was increased after low-dose drug and inhibited after high dose (2.5 mg/ml). Splenic CFU-Meg was reduced initially, followed by an increase at Week 4. Plasma titer of erythropoietin was elevated, proportional to dose escalation of drug, and inversely proportional to the hematocrit. No difference was observed in plasma levels of granulocyte-macrophage colony-stimulating factor, megakaryocyte colony-stimulating factor, or tumor necrosis factor-alpha. This study demonstrates that zidovudine-induced anemia results from: (i) inadequate numbers of bone marrow-derived, erythropoietin-dependent hematopoietic progenitors, i.e., CFU-E; and (ii) a shift in erythropoietin-responsive progenitors from bone marrow to spleen capable of responding to obligatory growth factors.     

Effect of 6-hydroxydopamine on murine hematopoietic stem cells: enhanced cytotoxicity on megakaryocyte colony forming units

We examined the effect of catecholaminergic neurotoxin 6-hydroxydopamine (6-OHDA) on murine committed megakaryocyte progenitor cells, the megakaryocyte-colony forming unit (CFU-Meg). More mature cells of the megakaryocyte series have the capacity for active uptake of catecholamines, and we speculated that the CFU-Meg would also take up 6-OHDA and be selectively killed. CFU-Meg were much more sensitive to the effects of this agent than were granulocyte-macrophage colony forming units (CFU-GM) or spleen-colony forming units. Co-incubation with catalase, which would destroy hydrogen peroxide generated extracellularly by the autoxidation of 6-OHDA, ablated 6-OHDA toxicity towards CFU-GM, but also significantly reduced the effect on CFU-Meg. Mazindol, a selective dopamine uptake inhibitor did not alter 6-OHDA effect on either CFU-Meg or CFU-GM. Finally, CFU-Meg were no more sensitive to incubation with hydrogen peroxide than were CFU-GM. These data suggest that CFU-Meg, unlike their more mature progeny, do not actively concentrate 6-OHDA, and the excess toxicity of this agent towards CFU-Meg is probably due to increased sensitivity to autoxidation products of 6-OHDA, other than hydrogen peroxide, generated extracellularly.     

Modulation of haematopoietic progenitor development by FLT-3 ligand.

The Flt-3 receptor is expressed in primitive haematopoietic cells and its ligand exerts proliferative effects on these cells in vitro in synergy with other cytokines. To increase our knowledge of the functional properties of the human Flt-3 ligand (FL) as relating to in vitro expansion of haematopoietic stem cells, the effects on murine haematopoiesis of FL alone or in combination with other growth factors were studied. Analysis of Flk-2/Flt-3 mRNA expression indicated that Flk-2/Flt-3 was preferentially expressed in primitive haematopoietic cell populations. To examine the expression of the Flk-2/Flt-3 receptor on megakaryocyte progenitors (CFU-Meg), Flk-2/Flt-3 positive and negative CD34(+)populations were separated from human bone marrow and cultured in a plasma clot culture system. CFU-Meg colonies were found in the Flk-2/Flt-3 negative fraction. Myeloid (CFU-GM) derived colonies appeared in the presence of FL alone. Neither FL+IL-3 nor FL+IL-3+IL-6 had any effect on the generation of megakaryocyte colonies (CFU-MK), due to the lack of FL receptor expression on megakaryocyte progenitors. Bone marrow cells remaining after 5-fluorouracil (5-FU) treatment of mice represent a very primitive population of progenitors enriched for reconstituting stem cells. This cell population expressed FL receptors, as revealed by RT-PCR analysis. Addition of FL alone did not enhance the replication of such cells in liquid cultures as compared to controls. However, a significantly greater generation of myeloid progenitors (CFU-GM) in clonogenic assays was observed in the presence of FL+IL-3, FL+GM-CSF or FL+CSF-1. In addition, the effects of FL on in vitro expansion of murine haematopoietic stem cells were studied using lineage-negative (lin(-)) Sca-1 positive (Sca-1(+)) c-kit positive (c-kit(+)) marrow cells from 5-FU treated mice. FL enhanced the survival of primitive murine lin(-)Sca-1(+)c-kit(+)cells. FL and IL-6 were able to significantly expand murine progenitor stem cells in vitro and promote their survival. These studies strongly suggest that FL significantly and selectively enhanced the generation of myeloid progenitors in vitro and increased myeloid progenitor responsiveness to later acting growth factors. In addition, FL synergized with IL-6 to support in vitro expansion of haematopoietic progenitors and promoted the survival of lin(-)Sca-1(+)c-kit(+)cells.     

Increased hematopoietic toxicity following administration of interferon-å with combination dideoxynucleoside therapy (zidovudine plus DDI) administered in normal mice

Because of the urgency to develop drugs which will effectively combat HIV infection, many combination therapies which have proved effective against HIV in vitro have undergone, or are undergoing clinical trial. Unfortunately many of drugs are being used without rigorous and exhaustive preclinical evaluation to assess their potential to develope hematopoeitic toxicity. We report here the results of two in vivo studies performed to analyze the effect of combined zidovudine (AZT) plus didanosine (ddl) therapy, either with or without interferon-å (IFN-å), on murine hematopoiesis. Normal C57BL/6 female mice were administered AZT (1.0 mg/ml) plus dose-escalation ddI (0.1, 1.0 and 2.5 mg/ml) placed in their drinking water. Control mice received IFN-å (100 units/ml) alone. Mice were serially bled and sacrified over a six-week period for assessment of hematopoietic toxicity measured by peripheral blood indices and assays of hematopoietic progenitors, i.e., erythroid (BFU-E), myeloid (CFU-GM), and megakaryocyte (CFU-Meg) cultured from bone marrow and spleen. AZT plus dose-escalation ddI decreased the hematocrit and white blood cell count when administered to normal mice compared to untreated controls during the six-week examination period. Marrow derived BFU-E, CFU-GM, and CFU-Meg were all reduced, however an increase was observed from the spleen for all three progenitor cell types. Use of IFN-å, in addition to combination AZT plus ddI further decreased the hematocrit, white blood cell and platelets. Marrow derived CFU-GM and CFU-Meg were increased slightly and only marginally for BFU-E with a similar response observed from the spleen. These results demonstrate that combination AZT plus ddI when used in vivo may produce synergistic hematopoietic toxicity, and that the addition of IFN-å to this treatment regimen increases this toxicity. These data indicate caution when this therapeutic approach is suggested for patients infected with HIV. If used, these patients wil require careful monitoring for blood cel toxicity.     

Stimulation of in vitro hematopoiesis by a murine fetal hepatocyte clone through cell—cell contact

We have previously shown that a fetal liver-derived epithelial cell clone, FHC-4D2, could support hematopoiesis in vitro through its colony-stimulating factor (CSF) activities in a short-term culture. In this study, since FHC-4D2 cells were found capable of maintaining hematopoietic progenitors in the coculture for a long time, we examined how FHC-4D2 could exert hematopoietic supporting activity in a long-term culture by coculturing adult bone marrow (BM) cells or fetal liver (FL) cells on a monolayer of FHC-4D2 cells. This clone could maintain the colony-forming unit of granulocytes and macrophages (CFU-GM) of BM for ≥ 12 weeks under the coculture condition, but the fibroblastic cell clone from the fetal liver, FHC-4A3, could not support the survival of CFU-GM, even for 1 week. In addition to BM CFU-GM, the FHC-4D2 clone also supported the survival of FL CFU-GM, burst-forming unit of erythroid cells (BFUe), and colony-forming unit of mixed progenitors (CFU-Mix) for longer than 4 weeks. When BM cells were separated by a membrane filter from the FHC-4D2 cells in the coculture, the comparable number of CFU-GM was maintained at day 3, but virtually no hematopoietic progenitors were detected at the end of the first week. CFU-GM were present in both nonadherent and adherent cells to the FHC-4D2 cells at day 3 of the coculture, but at day 7, the adherent population contained greater number of CFU-GM. CFU-GM derived from the adherent cells formed larger colonies and contained more bipotential CFU-GM than the nonadherent population. When BM cells from mice given 5-fluorouracil were cocultured with FHC-4D2 cells under the limiting dilution condition, interleukin-3 (IL-3)-responsive CFU-GM were induced from immature hematopoietic progenitor cells that were otherwise unresponsive to IL-3. From these data we conclude that the FHC-4D2 clone could generate and maintain IL-3-responsive hematopoietic progenitors via close contact and that, in the fetal liver, the contact between hepatocytes and hematopoietic cells may be critically important in inducing the differentiation of resting, IL-3-unresponsive immature hematopoietic cells into CFU-GM (progenitors responsive to IL-3) and in triggering the self-renewal of CFU-GM. © 1994 Wiley-Liss, Inc.     

kappa-opioids induce a reversible inhibition of CFU-GM from CD133(+) cord blood cells

BACKGROUND: Opioid agonists have been shown to exert an inhibitory action on a number of malignant and non-malignant cell types. However, there are no reports dealing with their effect on hemopoietic progenitors. Based upon our previous experience of opioid agonists we examined whether opioids could interfere with the growth of CFU-GM from CD133(+) cord blood cells. METHODS: Cord blood samples were subjected to CD133(+) column selection, with subsequent exposure to opioid agonists and antagonists or both, in semi-solid cultures for CFU-GM growth. Colonies of day 7 of culture were replated in fresh medium in the absence of opioids. The colonies were evaluated at 7 and 14 days of culture. RT-PCR was performed for the detection of opioid and somatostatin receptors. Apoptosis tests and immunophenotypic evaluations were employed in liquid cultures in conditions identical to those of the semi-solid ones. RESULTS AND DISCUSSION: Our results suggest that opioids can induce a significant inhibition of CFU-GM growth, which is reversible and not mediated through opioid or somatostatin receptors, while apoptosis is not implicated. Whether this finding could be used for clinical intervention remains to be examined.     

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.     

Positive and negative hematopoietic cytokines produced by bone marrow endothelial cells

Recently, cytokines and interleukins such as SCF, GM-CSF, G-CSF, TGF-beta, IL-6, IL-7, IL-8, IL-11 have been reported to be elaborated by endothelial cells. For further study, serum free bone marrow endothelial cell conditioned medium (BMEC-CM) was collected and ultrafiltrated by using a centriprep 10. The concentrated retentate (R-BMEC-CM) contained some substances whose molecular weight was more than 10 000 daltons. The filtrate (F-BMEC-CM) contained some substances whose molecular weight was less than 10 000 daltons. The effects of R-BMEC-CM and F-BMEC-CM on the growth of haematopoietic progenitors and the expression of cytokine and interleukin mRNAs of BMEC were investigated. The results showed that R-BMEC-CM stimulated the growth of CFU-GM, HPP-CFC, BFU-E, CFU-E, and CFU-Meg; while F-BMEC-CM inhibited the growth of these progenitors. Using the method of hybridizing to the Atlas cDNA Array, we were able to detect the presence of mRNAs of cytokines and interleukins in bone marrow endothelial cells. Our finding of the existence of mRNAs of SCF, GM-CSF, IL-6, TGF-beta, IL-1, and IL-11 in these cells was in agreement with the data reported previously. Furthermore, we detected mRNAs of MIP-2, Thymosion-beta4, PDGF, MSP-1, IFN-gamma, IL-13 and inhibin, which are related to haematopoiesis. Among these cytokines and interleukins, SCF, GM-CSF, IL-6, IL-1, and IL-11 are haematopoietic stimulators which may be responsible for the stimulative effects on the growth of haematopoietic progenitors. One of our new findings, the thymosin-beta4, is a small molecular haematopoietic inhibitor. It may be responsible for the inhibitory effect of F-BMEC-CM on haematopoietic progenitors. The presence of mRNAs of BMP, MSP-1, MIP-2, PDGF and IL-13 suggests that bone marrow endothelial cells might elaborate these substances. Their influence on haematopoietic progenitors needs further study.     

Demonstration of a protector effect of interleukin-1 against hematologic toxicity of azidothymidine (AZT)]

3'-azido-3'-deoxythymidine (Azidothymidine or AZT) has attained wide critical utility in the treatment of acquired immunodeficiency syndrome (AIDS). Unfortunately, treatment with AZT is associated with the development of severe hematopoietic toxicity. The AZT sensitivity of marrow progenitors was different with an IC 50 of 10(-8) M and 10(-6) M for respectively BFU-E and CFU-GM/GEMM. Data reported here show that recombinant human interleukin-1 alpha (IL-1 alpha), a pleiotropic cytokine, was demonstrated to be efficient to protect normal human as well as murine hematopoietic progenitors (CFU-GM, CFU-GEMM and BFU-E) from the toxic effect of AZT. The maximal effect was observed with 30 U/ml (Human cells) or 100 U/ml (murine cells) IL-1 alpha for BFU-E and CFU-GM/GEMM, with a marked effect at 1 U/ml. The results demonstrate that marrow progenitors respond differently to AZT and point out the potential efficacy of IL-1 alpha to enhance the proliferation of hematopoietic stem cells treated with growth factors (IL-3, erythropoietin) and to minimize the hematopoietic toxicity associated with AZT treatment.     

胎儿肝脏中一种抑制HL—60细胞生长的因子初步研究

胎儿肝脏中存在着两类抑制HL-60细胞生长的抑制物,一类是精氨酸酶,它是一类非特异性的细胞毒剂,在我们的实验条件下,不仅对HL-60细胞,而且对正常人骨髓CFU-GM也具有相似的抑制细胞生长的毒性作用。此外,还存在着一类较小分子的抑制物,它对HL-60细胞生长的抑制作用明显高于对人骨髓CFU-GM的作用,因此,在一定程度上,这是一类对HL-60细胞生长具有选择性作用的抑制物。     

In vitro activity of folic acid on the proliferation dynamics of human bone marrow CFU-GM in propyphenazone-induced granulocytopenia]

The in vitro action of folic acid was tested on the proliferation of bone marrow granulocyte-macrophage progenitor cells from a patient with drug-induced (propyphenazone) neutropenia in remission 20 days after the drug had been suspended. Various bone marrow cultures were prepared with standard stimulant, adding, respectively: folic acid, propyphenazone and both folic acid and propyphenazone together. Growth was tested on day 7, 12 and 19 of incubation. Under baseline culture with standard stimulant, CFU-GM growth was characterized by successive proliferation waves of various entity: the first on day 7 was very high, the second, on day 12 was rather low, and the third, on day 19 was intermediate. This behaviour is different from what is usually observed in normal subjects in steady-state, whose first (AC-A+AC-B) and second (AC-C) proliferation period are of similar entity. The prevalence of the first proliferation period in our case is interpreted as the result of a renewed granulocytopoietic activity after drug-induced bone marrow suppression. This indicates a maintained integrity of the negative-feedback mechanism of homeostatic regulation on granulocytopoietic activity. The sole addition of propyphenazone on the in vitro bone marrow cell cultures of our patient produced a reduction of those CFU-GM that had grown during the first period whereas the growth during the second and third period remained unvaried. Thus the growth peak in cultures treated with propyphenazone occurred on day 19, which seems to correspond with the necessary time for a spontaneous remission from neutropenia, clinically observed to be 20 days after suspension of the propyphenazone-containing drug.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Radiation sensitivity of megakaryocyte colony-forming cells in human placental and umbilical cord blood

The in vitro radiation sensitivity of CFU-Meg isolated from human placental and umbilical cord blood was evaluated in plasma clot cultures stimulated by recombinant human cytokines, including thrombopoietin, the FLT3 ligand (FLT3LG), interleukin-3, interleukin-11 and stem cell factor. The CD34(+) cells were irradiated with X rays at a dose rate of 73 cGy/ min. The megakaryocyte colonies were identified by using an FITC-conjugated antibody to glycoprotein IIbIIIa and were classified into two groups based on colony size: large colonies (immature CFU-Meg) and small colonies (mature CFU-Meg). Treatment with thrombopoietin alone or in combination with FLT3LG and/or interleukin-11 gave exponential radiation survival curves (D(0) for immature CFU-Meg = 56-77 cGy, D(0) for mature CFU-Meg = 86 cGy-1.12 Gy), while marked shoulders were observed on the survival curves for colonies supported by the combination of thrombopoietin, interleukin-3 and stem cell factor (D(0) for immature CFU-Meg = 89- 98 cGy; D(0) for mature CFU-Meg = 1. 25-1.31 Gy). Our results showed that the immature CFU-Meg were more radiosensitive than the mature CFU-Meg and that the combination of cytokines, including thrombopoietin, interleukin-3 and stem cell factor, affected the radiation sensitivity of CFU-Meg to the same extent as with thrombopoietin alone or in combination with FLT3LG and/or interleukin-11.     

Thy-1 is a differentiation antigen that characterizes immature murine erythroid and myeloid hematopoietic progenitors

To determine the role of Thy-1 antigen in murine hematopoietic differentiation, bone marrow was treated with anti-Thy-1.2 antibody and complement or complement alone. Growth of immature hematopoietic progenitors, erythroid burst-forming units (BFU-E), and granulocyte/macrophage colony-forming units (CFU-GM) was greatly reduced following antibody and complement treatment and was not restored by mitogen-stimulated spleen cell supernatants. In contrast, more mature erythroid and myeloid progenitors, the erythroid colony-forming unit (CFU-E) and the macrophage progenitor stimulated by L-cell-conditioned media (LCM), were spared by anti-Thy-1.2 antibody and complement treatment. Here, to separate the effects of anti-Thy-1.2 antibody treatment on accessory cells from those on progenitors, splenic T cells and thymocytes were added to treated marrow at ratios of up to 200%. Growth of BFU-E and CFU-GM was not restored. To more precisely replace required accessory cells, male complement-treated marrow was cocultured with female anti-Thy-1.2 antibody and complement-treated marrow. Even marrow cells failed to restore female BFU-E and CFU-GM growth. Fluorescent-activated cell sorting (FACS) and immune sheep red cell rosetting with anti-Thy-1.2-labeled marrow were then performed to determine if immature hematopoietic progenitors bear Thy-1.2. These techniques revealed enrichment of BFU-E and CFU-GM in the Thy-1.2-positive fraction, demonstrating the presence of Thy-1.2 on early murine hematopoietic progenitors. CFU-E and CFU-M were present in the Thy-1.2-negative fraction following FACS separation. These data demonstrate that Thy-1.2 is a differentiation antigen, present on at least some murine BFU-E and CFU-GM and lost as they mature to CFU-E and CFU-M.     

Effect of testosterone on the in vitro proliferation of bone marrow granulocyte-macrophage cells (CFU-GM). II. Observations in hypogonadal subjects

CFU-GM cultures in agar double layers were performed from bone marrow unfractionated cells of four subjects with hypogonadism, before and 24 hours after acute administration of 100 mg of testosterone propionate (Tp). Cell aggregates (CA) of CFU-GM were classified according to their sizes (P1 = 3-10 cells; P2 = 11-30; P3 = 31-50; P4 = over 50 cells) and according to their appearance time in culture (CA-A: appearing at the 7th day; CA-C: appearing only at the 12th day). The total of proliferating progenitors (tPP) also embraces CA present, in the same microscopic field, on both scoring times (CA-B). In all hypogonadal men studied, the treatment with Tp yielded increase of tPP (on the average of 65%) and increase of the total number of cells appeared in culture (TCP, increase on the average of 82%) calculated as product [CA number] x [average size of CA]. These results are in agreement with those already observed by us in CFU-GM cultures of normal subjects. Yet it is interesting to note that, while in normal subjects the exogenous testosterone effect expresses itself with a higher increase of CFU-GM number in the second interval of culture (CA-C), in hypogonadal men the CFU-GM number increase occurs mostly in the first period of the culture (CA-A).(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of SR-4987 stromal cells in the modulation of doxorubicin toxicity to in vitro granulocyte-macrophage progenitors (CFU-GM).

Bone marrow stromal microenvironment is essential for the maintenance of the hematopoietic stem cell renewal both by cell-cell interaction and cytokine production. However, stromal cells also exhibit drug metabolizing activities and they may accumulate the drug and successively affect hematopoietic progenitors by a retarded release. Our study investigated the role of both primary culture of murine bone marrow stroma and established stromal cells (SR-4987) in modulating the "in vitro" toxic activity of Doxorubicin (DXR) against murine granulocyte-macrophage progenitors (CFU-GM). The main part of the study has been performed by a "in vitro" agar bilayer technique based on the CFU-GM assay performed over a feederlayer of stromal cells. The results suggest that bone marrow stromal cells play also an important role in decreasing the toxicity of Doxorubicin. Further SR-4987 stromal cells produce a Doxorubicin metabolite (not belonging to the series of metabolites described in literature) which is completely ineffective in inhibiting the growth of CFU-GM and the activity of topoisomerase I. Our data suggest that bone marrow stromal cells must be considered as a cell population having opposite pharmacological roles in modulating the drug toxicity on hematopoietic progenitors. In our model a mechanism of detoxification concerns the capacity of SR-4987 stromal cells to inactivate the drug. For a better prediction of drug hematotoxicity, it is very important to develop "in vitro" cell models able to discriminate between positive and negative modulation of drug toxicity that stromal cells can exert in the bone marrow microenvironment.