Effects of recombinant human tumor necrosis factor (rhTNF) on normal human and mouse hemopoietic progenitor cells

We examined the effects of recombinant human tumor necrosis factor (rhTNF) on normal human and murine granulocyte-macrophage (CFU-gm) and erythroid (CFU-e, BFU-e) progenitor cells. We suppressed in vitro colony formation by human marrow CFU-gm, CFU-e and BFU-e or peripheral blood BFU-e by adding rhTNF to the culture in a dose-related manner. A half-maximal inhibition was observed with 1-10 ng/ml. Leukemic cell line K562 cells were found to be sensitive to rhTNF in the clonogenic colony assay. However, the clonal growth of murine marrow CFU-e and BFU-e colonies was less than 50% inhibited and CFU-gm growth was unaffected even at a concentration of 1,000 ng/ml. We observed slight to moderate inhibition after 24 h pulse exposure of both human and murine-committed progenitors to rhTNF prior to the culture. Intravenous injection of 1 mg/kg of rhTNF caused a marked decrease in marrow erythroid progenitors and consequently caused anemia in the mice. Our data indicate that rhTNF has a suppressive effect on normal human and murine hemopoietic colony formation in vitro and murine erythropoiesis in vivo.     

Effects of dolichol and dolichyl phosphate on in vitro differentiation of hematopoietic progenitors

The exogenous addition of dolichyl phosphate (Dol-P), an active form of dolichol (Dol) that carries oligosaccharide chains for protein-N-glycosylation, significantly enhanced colony formation of mouse bone marrow hematopoietic progenitors (CFU-e, BFU-e, and CFU-gm) was stimulated by erythropoietin (Epo) and colony-stimulating factor (CSF), but Dol enhanced colony formation of CFU-e only. The effects of Dol or Dol-P on these hematopoietic progenitors were fully dependent on stimulation by Epo or CSF. Other mevalonate-metabolites, such as cholesterol, coenzyme Q10, and isopentenyladenine, had no effect on hematopoietic progenitors. These studies suggest that exogenous Dol-P enhances the frequency of differentiation of hematopoietic progenitors stimulated by Epo or CSF, and there may be a diversity in cellular response of these progenitors to Dol.     

The effect of carbamylcholine on CFU-s differentiation

The proportion of spleen colony-forming units (CFU-s) killed by hydroxyurea was greatly increased after bone marrow cells (BMCs) from LACA mice were exposed to carbamylcholine (Cach; 1 X 10(-13) to 1 X 10(-9) in vitro and there was a marked change in the proportion of spleen colony types. Following treatment with Cach, granulocytic and mixed erythroid-type colonies increased from 20 to 26.3% and 16.1 to 29.6% in 9-day colonies and from 8.3 to 28.2% and 21.7 to 39.4% in 13-day colonies, respectively. Single cell suspensions of spleen colonies were made for granulocyte-macrophage progenitor (CFU-gm) and late erythroid progenitor (CFU-e) assays. The number of CFU-gm from Cach-treated BMC was about twice that from control BMC for both day 9 and day 13 groups; the number of CFU-e decreased relatively. The results suggest that cholinergic receptors on CFU-s may increase the tendency to differentiate into the granulocytic/monocytic line.     

Acquisition and clonal growth of human sternal bone marrow obtained incidental to open-heart surgery

The establishment of a convenient method of acquiring ample human bone marrow and the successful growth of hematopoietic precursor cells in standard methylcellulose assay is described. Human sternal marrow curettings were obtained at open-heart surgery. Growth of colonies derived from CFU-gm, BFU-e, and CFU-e was successful. Comparison of the CFU-gm colony production between this and prior studies indicates a consistent mean and range of growth. These results document this to be an efficient source of normal human marrow.     

Microcapillary clonogenic assays for human marrow hematopoietic progenitor cells

The capillary clonogenic cell assay was developed and adapted to culture myeloid and erythroid colonies from human bone marrow cells. The plating efficiencies for femoral bone marrow granulocyte-macrophage progenitors (CFU-gm), erythroid colony-forming units (CFU-e) and erythroid burst-forming units (BFU-e) were 0.143%, 0.229% and 0.141%, respectively. Standard bone marrow progenitor Petri dish assays require a total culture volume of 1 ml per dish, and as such are not suitable for the small numbers of cells often obtained from human bone marrow samples. The microcapillary assay as developed and standardized in our laboratory has the unique advantage of being able to utilize small numbers of cells. This technique is suitable for evaluating the myelotoxicity of investigational new anti-cancer and anti-HIV agents and for further investigation of the mechanisms underlying chemotherapy-induced bone marrow toxicity.     

Kinetics of the Hb A to Hb C switch and erythropoietin plasma levels in sheep

The induction of Hb A (alpha 2 beta A2) and Hb C (alpha 2 beta C2) synthesis in three adult sheep has been sequentially analysed, in relation to the reduction of the haematocrit (Ht) and to the changes of erythropoietin (Epo) concentration in plasma. Hb A production is detected in peripheral reticulocytes when the Ht approaches 70% of its initial value in correspondence with the first rise of EPO plasma level, whereas HB C synthesis appears when the Ht is further reduced to about 50%, at an Epo concentration two to three times higher. The assumption that the cell committed to HB C synthesis is close to the erythroid colony-forming unit (CFU-e) progenitor is also discussed.     

Accelerated marrow recovery following total-body irradiation after treatment with vincristine, lithium or combined vincristine-lithium

Accelerated post-irradiation recovery of hematopoietic marrow has been reported following treatment with lithium (Li) or vincristine (VcR). Because these two agents appear to exert their effects on different, albeit overlapping, hematopoietic populations, it was felt that combining them might lead to a wider spectrum of enhanced post-irradiation marrow regeneration. Results demonstrated that an accelerated recovery, which appeared to be additive in nature, was observed in the marrow following combined VcR-Li/4.5 Gy total-body irradiation. The combined schedule significantly enhanced post-irradiation recovery of white blood cells, 12-day spleen colony-forming units, erythroid burst-forming units, and fibroblastic colony-forming units over radiation alone; and recovery of marrow cellularity, multipotential colony-forming units (CFU-gemm) and granulocytic/monocytic colony-forming units (CFU-gm) over both radiation alone and either drug given singly with the 4.5 Gy. In addition, while data on the ability of regenerating stroma to support CFU-gm and CFU-gemm did not suggest that VcR was acting to enhance post-irradiation marrow recovery by increasing stromal production of hematopoietic growth factors, Li did appear to increase production of one or more of these factors, and this may be part of its mechanism of action.     

CSF-responsive bone marrow cells in liquid culture: characterization and screening applications

In a previous study, colony-stimulating factor (CSF) activity assayed in colony culture correlated closely with 3HTdR uptake by human marrow cells depleted of adherent cells. To use this assay for screening media for CSF and immunotoxins for marrow toxicity, cells growing in liquid culture were compared to conventional granulocyte/macrophage (CFU-gm) colony assays. CSF dose-response relationships for liquid and colony-forming assays were nearly identical. 3HTdR uptake by nonadherent marrow cells was CSF dose-related, and there was a linear relationship between number of cells cultured and 3HTdR uptake. Ricin cytotoxicity curves for liquid cultures and CFU-gm were identical on day 7 but showed some disparity with day 14 cultures. Results with all cultures showed 3HTdR uptake to be most closely correlated with CFU-gm colony, rather than cluster, growth. Myeloid cell differentiation in liquid culture was similar to colony cultures, producing mixtures of granulocytes, macrophages and eosinophils. By combining cell and differential counts, production of various myeloid cells could be quantitated. Cytotoxicity of anti-Ia for CFU-gm and liquid culture cells was compared and the majority of both cell populations expressed Ia-like antigens. Simultaneous staining for surface antigens and DNA content was used to characterize proliferating marrow cells, and the vast majority of cells expressed myeloid markers. Transferrin receptors were displayed by cells in S/G2/M and appeared after CSF stimulation on G0/G1 cells. We conclude liquid cultures can be used to screen conditioned media for human CSF and to screen for cytotoxicity to normal myeloid precursor cells. Behavior of CSF-responsive cells in liquid culture appears most closely related to that of CFU-gm colony-forming cells, and characterization of CSF-stimulated cells allows quantitative as well as qualitative estimates of myeloid cell production.     

Effects of recombinant interferons on the clonogenic growth of leukemic cells and normal hemopoietic progenitors

We have examined the effects of recombinant immune and leukocyte interferons (rIFN-gamma and rIFN-alpha) on the clonogenic growth of leukemic cells and normal hemopoietic progenitors using in vitro colony assays. Both interferons suppressed the colony formation by granulocyte-macrophage progenitors (CFU-gm) and erythroid progenitors (CFU-e and BFU-e) in a dose-dependent manner. Six myeloid leukemic cell lines were less sensitive to rIFN-gamma than CFU-gm. The colony formation of some myeloid leukemic cell lines was suppressed more potently by rIFN-alpha than by CFU-gm. Four lymphoid leukemic cell lines of the T-cell type were very resistant to both recombinant interferons. Reduced sensitivity of leukemic cells to rIFN-gamma, a possible hemopoietic regulator, may explain partially the unregulated proliferation of leukemic cells in vivo.     

Characterization of the human erythroid progenitor cells responsive to the erythropoietic-enhancing activity found in normal human serum

Normal human serum significantly increased the growth of erythroid colonies from bone marrow colony-forming units-erythroid (CFU-e) which were enriched by using a set of monoclonal antibodies in a panning technique. This activity was still observed in cultures of enriched CFU-e plated near the limiting cell dilution. When the addition of erythropoietin was delayed so that only early CFU-e could survive, we observed that the growth of erythroid colonies was less affected in cultures containing erythropoietin and normal serum than in those containing erythropoietin only. We have concluded from this study that normal human serum acts on in vitro erythropoiesis by directly stimulating the growth of a population of early CFU-e.     

Comparative distribution of marrow CFU-e and CFU-gm progenitors in different anatomic sites in the dog

The interpretation of marrow cloning activity, particularly in serial cultures, is greatly influenced by the reproducibility of the collected marrow samples. In order to establish whether bone marrow cloning activities and precision of the cloning assays are influenced by the site of bone marrow collection in the dog, we studied the incidence of marrow erythroid (CFU-e) and granulocyte-macrophage (CFU-gm) progenitor cells in the iliac crest, sternum, vertebrae, femur, and humerus, using microplasma clot and soft agar culture systems. Marrow samples obtained from the femur and humerus revealed consistently higher cell concentrations than those from the iliac crest, vertebrae, or sternum. Those aspirated from the sternum and vertebrae had lower cell concentrations and were less reproducible. Statistical analysis revealed no significant differences in the incidence of marrow CFU-e and CFU-gm progenitor cells between the femur, humerus, iliac crest or vertebrae. With multiple sampling, the marrow cloning efficiency was consistent and reproducible within the individual dogs. We conclude that the distribution of CFU-e and CFU-gm is comparable throughout the active marrow in the dog and that these sites may be used interchangeably for multiple quantitative analysis of marrow hematopoietic progenitor cells.     

The enhancement of committed hematopoietic stem cell colony formation by nandrolone decanoate after sublethal whole body irradiation

The ability of an anabolic steroid, nandrolone decanoate, to increase committed topoietic stem cell (CFU-gm, CFU-e, and BFU-e) colony formation after sublethal irradiation was evaluated. Immediately after receiving whole body irradiation and on the next two days, each mouse was injected intraperitoneally with nandrolone decanoate (1.25 mg) in propylene glycol. Irradiated control mice received only propylene glycol. Compared to controls, drug-treated mice showed marked peripheral blood leukocytosis and more stable packed red cell volume. Drug-treated mice also demonstrated increased erythropoiesis, as CFU-e/BFU-e concentrations from both marrow (9% to 581%) and spleen (15% to 797%) were elevated. Granulopoiesis was increased similarly, as CFU-gm concentrations from marrow (38% to 685%) and spleen (9% to 373%) were elevated. These results demonstrate that nandrolone decanoate enhances hematopoietic stem cell recovery after sublethal whole body irradiation. This suggests that following hematopoietic suppression, nandrolone decanoate may stimulate the recovery of hematopoiesis at the stem cell level and in peripheral blood.     

Improvement of culture conditions for human megakaryocytic and pluripotent progenitor cells by low oxygen tension

The effect of low oxygen tension on the growth of human hemopoietic progenitor cells in bone marrow was investigated using the semisolid methylcellulose colony assay. The clonal growth of granulocyte-macrophage progenitors (CFU-gm), early (BFU-e) and late (CFU-e) erythroid progenitors, megakaryocyte progenitors (CFU-meg) and pluripotent progenitors (CFU-mix) improved more markedly incubation at the low oxygen tension (5%) than in conventional air (20%). The thiol compound 2-mercaptoethanol had a strong additive effect on colony growth in conventional air, but little or no effect in the low oxygen tension. These results suggest that enhancement of colony growth in the low oxygen tension may be due to a decrease in the production of oxygen intermediates.     

Effect of recombinant human interleukin 3, granulocyte-macrophage colony-stimulating factor and granulocyte colony-stimulating factor on human BFU-e in serum-free cultures

The effects of recombinant human hemopoietic growth factors on early and late human erythroid progenitors (BFU-e and CFU-e) were investigated in serum-free cultures. Recombinant human erythropoietin (rhEpo) induced the formation of not only human CFU-e-derived colonies but also human BFU-e-derived bursts. Recombinant human interleukin 3 (rhIL-3) alone did not induce the formation of human BFU-e-derived bursts and human CFU-e-derived colonies. In the presence of rhEpo, rhIL-3 dose dependently increased the number of bursts stimulated by rhEpo, although rhIL-3 did not have the augmentative effect on human CFU-e growth. On the other hand, rhIL-3 did not stimulate the formation of murine BFU-e-derived bursts, and murine IL-3 did not stimulate the formation of human BFU-e-derived bursts. The results indicated that the burst-promoting activity of IL-3 was species-specific between human and murine cells. Recombinant human GM-CSF (rhGM-CSF) or recombinant human G-CSF (rhG-CSF) failed to induce human burst formation and did not augment the effect of rhEpo on human burst formation. The results of the present study suggest that in vitro, IL-3 can stimulate BFU-e in collaboration with Epo, but GM-CSF and G-CSF do not stimulate BFU-e growth in the presence or absence of Epo.     

Canine BFU-e progenitors: adaptation of a reproducible assay and anatomical distribution.

In vitro cloning assays are used increasingly in investigative hematotoxicology and in screening candidate compounds for their hematotoxic potential. To expand these applications, a practical cloning assay for erythroid burst-forming units (BFU-e) that uses a microplasma clot (MPC) system was adapted to the dog, a species used extensively in experimental hematology and drug development. This system offers the advantage over the methylcellulose and soft agar culture systems of allowing specimen fixation and, therefore, morphological and cytochemical evaluation. The distribution of BFU-e among various anatomic sites was assessed using the MPC cloning system, which was modified to optimize the BFU-e growth. BFU-e growth required only erythropoietin (Epo) in the culture medium and there was no need for an exogenous source of burst-promoting activity (BPA). The cloning efficiency was linearly proportional to the plating concentrations of Epo and marrow mononuclear cells (MMC) over a range of 0 to 3 U Epo and 1 x 10(5) to 3 x 10(5) MMC per ml of culture, respectively. Increases in concentrations of Epo and MMC beyond these levels were not associated with linear growth. The addition of transferrin and spleen-conditioned medium containing a mixture of growth factors (including BPA) reduced BFU-e growth. The relative concentration of BFU-e was comparable among samples collected from the iliac crest, femur, and humerus. Serial cultures performed on individual dogs were highly reproducible and there was little variation in BFU-e activity among dogs of comparable age. It was concluded that the MPC system is a practical and reproducible cloning system for early (BFU-e), as well as late erythroid colony-forming units (CFU-e) in the dog. The concentration of BFU-e appears comparable throughout the active marrow; therefore, various anatomic sites can be used interchangeably for serial quantitative analysis of this progenitor.     

VEGF modulates erythropoiesis through regulation of adult hepatic erythropoietin synthesis

Vascular endothelial growth factor (VEGF) exerts crucial functions during pathological angiogenesis and normal physiology. We observed increased hematocrit (60-75%) after high-grade inhibition of VEGF by diverse methods, including adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains, recombinant VEGF Trap protein and the VEGFR2-selective antibody DC101. Increased production of red blood cells (erythrocytosis) occurred in both mouse and primate models, and was associated with near-complete neutralization of VEGF corneal micropocket angiogenesis. High-grade inhibition of VEGF induced hepatic synthesis of erythropoietin (Epo, encoded by Epo) >40-fold through a HIF-1alpha-independent mechanism, in parallel with suppression of renal Epo mRNA. Studies using hepatocyte-specific deletion of the Vegfa gene and hepatocyte-endothelial cell cocultures indicated that blockade of VEGF induced hepatic Epo by interfering with homeostatic VEGFR2-dependent paracrine signaling involving interactions between hepatocytes and endothelial cells. These data indicate that VEGF is a previously unsuspected negative regulator of hepatic Epo synthesis and erythropoiesis and suggest that levels of Epo and erythrocytosis could represent noninvasive surrogate markers for stringent blockade of VEGF in vivo.     

Altered colony-forming activities of bone marrow hematopoietic stem cells in mice following short-term in vivo exposure to parathion

This paper describes a study of hematopoiesis in parathion-treated mice. Adult mice (48 C57B1/6) were given a daily dose of parathion (4 mg/kg p.o.) or corn oil vehicle (5 ml/kg p.o.) for 14 days. During the pesticide and the examination period, treated animals showed no signs of poisoning and had normal body weights. On days 2, 5, 7, 9, 12 and 14 following parathion or corn oil, femoral marrow cells were assayed in vitro for granulocyte/monocyte (CFU-gm), erythroid (CFU-e and BFU-e), megakaryocyte (CFU-meg), stromal (CFU-str) and multipotential (CFU-mix) hematopoietic stem cells. Leukocyte counts were elevated on days 2 and 5, while platelet counts were not increased until day 12. No change was observed in either hematocrits or numbers of marrow cells. BFU-e were reduced (23% of control) by day 7, then increased to 137% of control by day 14. CFU-e were reduced (41% of control) on day 9, then increased to 71% of control by day 14. CFU-mix were 130% of control (day 2), then declined to control values by day 5. On days 12 and 14, CFU-mix colonies decreased to 40% of control. CFU-str were reduced at all time points examined. CFU-gm were 123%, 136% and 130% of control on days 7, 12 and 14, respectively, while CFU-meg were increased (145% of control) on day 7. The data suggest that parathion alters the cloning potential of bone marrow precursor stem cells.     

Inhibition of erythropoietin production by phorbol ester is associated with down-regulation of protein kinase C-alpha isoenzyme in hepatoma cells.

The role of protein kinase C (PKC) in the control of erythropoietin (Epo) production was studied using the human hepatoma cell line HepG2. Inhibition of PKC by staurosporine and the selective PKC inhibitor CGP 41251 significantly reduced Epo formation. No inhibition occurred with the inactive staurosporine derivative CGP 42700. Treatment with phorbol 12-myristate 13-acetate (PMA) for 24 h dose-dependently inhibited Epo formation, thus suggesting that down-regulation of PKC might be responsible for this inhibition. Immunoblotting experiments showed that incubation of HepG2 cells with PMA for 24 h resulted in a selective and almost complete down-regulation of PKC-alpha. Thus, PKC-alpha may play a permissive role in Epo synthesis in HepG2 cells.     

Hemopoietic progenitor cell function after HLA-identical sibling bone marrow transplantation: influence of chronic graft-versus-host disease

We examined hemopoietic reconstitution during the first 12 months post-transplant in 31 patients given high-dose cyclophosphamide, total body irradiation and an HLA-identical sibling marrow transplant for hematological malignancy. Unexpectedly, we found marrow CFU-gm and marrow CFU-e cells to be denser than normal throughout the first year post-transplant. While functionally adequate neutrophil and platelet counts were achieved in the first six weeks post-transplant, there were defects in hemopoietic progenitor cell function during the first year post-transplant. Although we could detect no influence from acute graft-versus-host disease (GVHD), chronic GVHD adversely affected the growth of both myeloid and erythroid blood progenitor cells.     

CFU-gm colony formation of peripheral blood and bone marrow in adult acute leukemia at presentation, during remission, and at relapse

Granulocyte/macrophage colony-forming unit (CFU-gm) formation was studied simultaneously in bone marrow and peripheral blood of 52 previously untreated adult patients with acute non-lymphocytic (ANLL) and 36 with acute lymphoblastic leukemia (ALL). They were followed during induction therapy at monthly intervals while in remission and in 19 ANLL and 22 ALL cases, until relapse. Patients showing a decreased colony number in the marrow but normal or increased colony numbers in the peripheral blood had a high probability of entering remission. Non-responding patients displayed an opposite pattern. The higher the degree of marrow repopulation with granulocytic progenitor cells after induction treatment, the longer remission duration and survival for ANLL patients and the longer survival for ALL patients. CFU-gm formation returned to normal in the early stages of complete remission, but then declined progressively. At ANLL and ALL relapse, colony growth was reduced markedly while cluster formation remained normal. The number of marrow colonies and clusters in ANLL were significantly higher at first and second relapse compared to the growth pattern at first presentation. A similar trend had been observed in ALL, suggesting a selection advantage.