Comparison of hemin enhancement of burst-forming units-erythroid clonal efficiency by progenitor cells from normal and HIV-infected patients.

The ability of peripheral-blood hematopoietic progenitor cells from AIDS patients and normal controls to respond to erythropoietin (Epo) was assessed for burst-forming units-erythroid (BFU-E). BFU-E colony formation from AIDS patients' peripheral blood responded to a wide range of Epo concentrations (0.5-4 U) in a similar manner as erythroid progenitors obtained from normal peripheral blood. The optimum dose response of BFU-E to Epo was 2 U which resulted in generation of 71 +/- 4 BFU-E in AIDS patients (n = 10), as compared to 77 +/- 5 BFU-E in normal donors (n = 3). The optimum concentration range of hemin enhancement of erythroid progenitor BFU-E was 10-50 microM. In all instances, Epo was essential for BFU-E growth. Inclusion of hemin at a concentration of 10 microM in AIDS patients' peripheral-blood erythroid progenitor cells resulted in enhancement of BFU-E by 136-215%. Similarly, inclusion of hemin (10-100 microM) in normal bone marrow erythroid progenitor cell cultures resulted in enhancement of BFU-E. Inclusion of an equivalent amount of iron or tin protoporphyrin to progenitors cells from AIDS patients' peripheral blood had no effect on the number of colonies observed. On the other hand, inclusion of another heme analogue, zinc protoporphyrin, in AIDS or normal cultures resulted in a 50% suppression of BFU-E colony formation. These results demonstrate that peripheral-blood mononuclear cells from AIDS patients retain the capacity to generate erythroid precursors such as BFU-E in the presence of Epo, and that hemin has a specific enhancement effect on growth of BFU-E colony formation obtained from peripheral blood or bone marrow cells.     

Effects of recombinant human interferon gamma on hematopoietic progenitor cell growth

Human bone marrow BFU-E, CFU-E, and CFU-GM were cultured in the presence of varying concentrations of recombinant human interferon gamma (rHuIFN-gamma). Concentration-dependent inhibition of both erythroid and myeloid precursors by rHuIFN-gamma was demonstrated. A more pronounced suppressive effect of rHuIFN-gamma was seen on the BFU-E than on the CFU-E, with CFU-GM most resistant. rHuIFN-gamma was also added at varying time points during the marrow cultures, demonstrating different time-dependent sensitivities to rHuIFN-gamma; CFU-E were no longer sensitive to rHuIFN-gamma by day 2 of culture, BFU-E by day 6, and CFU-GM by day 9, indicating a loss of sensitivity with maturation. Finally, exposure of marrow cells to rHuIFN-gamma for varying periods of time prior to initiation of hematopoietic cultures failed to inhibit erythroid colony growth in the absence of rHuIFN-gamma in the culture. These studies demonstrate a suppressive effect of rHuIFN-gamma on human erythroid and myeloid progenitor cell growth. This effect appears to be most pronounced on the more primitive stages of committed progenitor cell development.     

Appearance of adherent cells suppressive to erythropoiesis during an early stage of Plasmodium berghei infection in mice.

We examined the effect of adherent cells from bone marrow or spleen of mice infected with Plasmodium berghei on dyserythropoiesis. Significant reduction in number of erythroid progenitors (erythroid colony-forming units: CFU-E and erythroid burst-forming units: BFU-E) was observed in bone marrow as early as 1 day after P. berghei infection. When adherent cells were removed from bone marrow or spleen cells of infected mice, the number of CFU-E and BFU-E was clearly increased. Furthermore, addition of adherent cells from infected mice to nonadherent cells from normal mice inhibited erythroid colony formation significantly in a dose-dependent manner. These results suggest that the adherent cells obtained from bone marrow or spleen of mice in the early stage of P. berghei-infection have a suppressive effect on erythropoiesis.     

Hemin stimulation of hemopoiesis in murine long-term bone marrow culture.

The effect of various concentrations of exogenous hemin on cellularity and hemopoietic clonal potential of cells maintained in murine long-term marrow cultures (LTBMC) was studied. Hemin, at concentrations of 1 and 10 microM, was added weekly to LTBMC and was found to produce a significant increase in cellularity for up to 8 weeks in culture. Lower concentrations of hemin (0.1 microM) were more effective for sustained cellularity in older cultures (10-12 weeks). Prior exposure of the adherent cell layer to high concentrations of hemin (10 microM) was found to have a beneficial effect on the support of newly seeded cultures; however, the effect of lower hemin concentrations (0.1-1 microM) on stromal cell layer formation was not significant. Supplementation of hemin for the first week in culture increased cumulative cell production as well as the number of granulocyte-macrophage colony-forming units (CFU-GM), and longevity of hemopoiesis in LTBMC was significantly increased with 0.1 microM hemin. In contrast with data obtained in short-term cultures, hemin in this system primarily affected the myeloid line of differentiation, whereas there was a less noticeable effect on the early erythroid progenitors (erythroid burst-forming units, BFU-E). Hemin, at 0.1 microM, increased spleen colony-forming units (CFU-S) to numbers several-fold higher than those of the control. Results suggest that hemin may produce mobilization of hemopoietic cells and committed precursors from adherent cells into suspension. Further, supplementation with hemin in LTBMC significantly increased the myeloid progenitor compartment and longevity of culture without altering the erythroid compartment.     

Interleukin-11 stimulates multiple phases of erythropoiesis in vitro.

Interleukin-11 (IL-11), a pleiotropic cytokine originally isolated from a primate bone marrow stromal cell line, has been shown to stimulate T-cell-dependent B-cell maturation, megakaryopoiesis, and various stages of myeloid differentiation, but to inhibit adipogenesis. Because stromal cells are essential for the maintenance of early hematopoietic progenitor cells in long-term culture, we investigated the effects of IL-11 on multipotent and erythroid precursors from murine bone marrow in vitro in suspension and semisolid cultures. Our results show that in the presence of IL-3 or c-kit ligand (KL), IL-11 has profound stimulatory effects on primitive multilineage hematopoietic progenitors, pre-CFC(multi), as well as on precursors representing various stages of erythroid differentiation observable in vitro, including CFC(multi), BFU-E, and CFU-E. In addition, the combination of KL with IL-11 also stimulated highly proliferative erythroid progenitors that yield remarkable macroscopic erythroblast colonies in culture. These results indicate that IL-11 is likely to play a pivotal role in early hematopoiesis and at multiple stages of erythropoiesis.     

Comparative influences of phytohemagglutinin-stimulated leukocyte conditioned medium, hemin, prostaglandin E, and low oxygen tension on colony formation by erythroid progenitor cells in normal human bone marrow

The comparative influences of phytohemagglutinin-stimulated leukocyte conditioned medium (PHALCM), hemin, prostaglandin E1 (PGE1), and growth of cells at low oxygen tension (5% O2) were evaluated for their capacity to enhance colony formation in vitro from normal human bone marrow erythroid progenitor cells (BFU-E). Each treatment enhanced colony formation by itself, and the combinations of treatments resulted in an additive enhancing effect on erythroid colony formation. Removal of T-lymphocytes from the bone marrow sample ablated the enhancing activity of PGE1, but did not influence the enhancing activities of PHALCM, hemin, and growth at low oxygen tension. The results suggest that the mechanisms of action of these various erythroid colony-enhancing effects may be different.     

The effect of estrogens on erythroid stem cells in polycythemic mice

Plethoric mice treated with pharmacological doses of estradiol have decreased concentration of erythropoietin-responsive cells (ERC) in the marrow. We used the methylcellulose-culture system for growth of erythroid stem cells (CFU-E and BFU-E) to define more accurately these estrogen-induced changes. As an animal model we utilized plethoric mice given repeated injections of estradiol cypionate and found that at 14 days after the onset of treatment there was no significant change in the concentration of femoral CFU-E whereas there was a significant decrease of the BFU-E content. Both CFU-E and BFU-E increased progressively in the spleen over a 42-day period. Addition of estradiol directly to the cell-culture system showed no effect on CFU-E growth but induced a significant depression of BFU-E growth. This depression seemed to require the presence of adherent cells. It is our hypothesis that estrogens suppress only the early stages of erythroid proliferation and/or differentiation by a mechanism involving possibly the stromal (adherent) cells of the marrow microenvironment.     

Enhancement of in vitro erythropoiesis by peripheral blood mononuclear cells from allogeneic marrow recipients in the early post-transplant period

The effect of peripheral blood mononuclear cells (PBMCs) from bone marrow recipients on in vitro growth of erythroid burst-forming units (BFU-E) was studied. PBMCs were obtained from 5 allogeneic, 1 syngeneic and 1 autologous bone marrow recipient(s) at different intervals after transplantation. The number of BFU-E was significantly increased when donor bone marrow cells were co-cultured with PBMCs obtained from allogeneic marrow recipients in the early post-transplant period. No effect was observed using PBMCs obtained in the later post-transplant period, PBMCs from a syngeneic marrow recipient, or PBMCs from an autologous marrow recipient. The BFU-E enhancing activity was present among T cells and was abolished by treating them with OKT3 or OKT4 antibody and complement. These observations suggest that chimeric T lymphocytes, probably of the helper/inducer subset, from allogeneic marrow recipients in the early post-transplant period have a potent enhancing effect on in vitro erythropoiesis.     

Further characterization of the hemin-induced enhancement of primitive erythroid progenitor cell growth in vitro

We have previously demonstrated that hemin specifically enhances the in vitro plating efficiency of primitive murine erythroid progenitors (day 7 BFUE), whereas it does not appear to affect more mature progenitors (mature BFUE or CFUE). In this report, we further characterize the effects of hemin on marrow-derived day 7 BFUE growth in vitro. BFUE were enhanced by hemin in a dose-dependent manner and to a greater extent in methyl cellulose than in plasma clot cultures. That hemin might increase the rate of cell division was suggested by the greater size of colonies grown in its presence as well as their earlier appearance in culture. In contrast, the addition of hemin to marrow cell cultures did not appear to affect the survival rate of BFUE or their progeny. While significantly augmenting the frequency of BFUE, hemin had no consistent stimulatory effect on CFUGM. Lastly, hemin was equally capable of augmenting burst growth in adherent cell-depleted as in whole marrow cell preparations. These experiments suggest that hemin augments directly and in a cell-specific manner the proliferation and/or differentiation of primitive marrow erythroid progenitors in vitro.     

Regeneration of erythroid progenitor cells in polycythemic mice treated with cyclophosphamide

Mice with posthypoxic polycythemia treated with a sublethal dose of cyclophosphamide (Cy) were used as a model to investigate, by in vitro methods, the kinetics of regeneration of erythroid committed precursors (ECP) and to study the influence of erythropoietin (Ep) on those precursor cells. The results demonstrated that erythroid burst-forming units (BFU-E), early (d10) and late (d4), and erythroid colony-forming units (CFU-E) recover at different rates after Cy. Early BFU-E recovery was not Ep dependent and closely resembled regeneration of pre-erythropoietin-responsive cells (pre-ERC) found previously using the same experimental model. The absence of spontaneous recovery of mature BFU-E and CFU-E in the bone marrow and spleen of Cy-treated polycythemic mice, which is contrary to the findings in normal mice treated with Cy, indicates the importance of Ep for BFU-E (d4) and CFU-E regeneration. This was confirmed when exogenous Ep was injected. The effect on BFU-E (d4) of exogenous Ep injected into the polycythemic Cy-treated mice at the time when primitive BFU-E have regenerated considerably suggested an influence of Ep on the transition of BFU-E (d10) to BFU-E (d4). The fast regeneration of CFU-E in the spleen of normal mice and after Ep injection in polycythemic Cy-treated mice confirms the well-known and significant role of the spleen in mouse erythropoiesis under stress conditions. It could be suggested that the patterns of BFU-E (d4) and CFU-E recovery as well as Ep responsiveness closely resemble the findings observed earlier for ERC in the same experimental model.     

Erythroid progenitors in polycythemia vera demonstrate a different response pattern to IL-4 compared to the normal BFU-E from peripheral blood

Human recombinant interleukin 4 (IL-4) was studied for its effects on erythroid burst-forming units (BFU-E) from normal peripheral blood and from patients with polycythemia vera (PV). IL-4 enhanced the proliferation of normal peripheral blood BFU-E (183% +/- 20% enhancement), whereas in the presence of interleukin 3 (IL-3) no further augmentation was noticed. The IL-4-mediated effects were independent of the absence or presence of adherent cells, B cells, or T cells. These data are in contrast with results obtained from normal human bone marrow cells, in which IL-4 antagonized the enhancing effects of IL-3. In PV a different response pattern was observed. The effects of IL-4 on the erythropoietin (Epo)-independent BFU-E were variable. In five PV patients no suppressive or enhancing effects of IL-4 were observed, whereas in two additional patients a significant decline in the Epo-independent BFU-E was noted. In the presence of IL-3, IL-4 significantly antagonized the IL-3-supported Epo-independent BFU-E in all patients (272% +/- 57% vs 187% +/- 49% enhancement, p less than 0.05). In contrast, IL-4 did not modify the IL-3-supported Epo-dependent BFU-E. In summary, these data suggest a difference between the normal and PV peripheral blood BFU-E. The Epo-dependent erythroid progenitors in PV patients showed a response pattern with IL-3 and IL-4 comparable to that of normal peripheral blood BFU-E, whereas the Epo-independent erythroid progenitors behaved like normal human bone marrow BFU-E, suggesting a shift in the stem cell compartment in PV. This is further supported by the finding that erythroid colony-forming units (CFU-E), normally only present in the bone marrow, could be cultured from the peripheral blood of PV patients in the presence or absence of Epo.     

Enhancement of the proliferation of human marrow erythroid (BFU-E) progenitor cells by prostaglandin E requires the participation of OKT8-positive T lymphocytes and is associated with the density expression of major histocompatibility complex class II antigens on BFU-E

The relationship between major histocompatibility complex class II antigens (MHC class II, eg, HLA-DR, Ia), T lymphocytes, and the enhancement of erythroid colony formation from BFU-E by prostaglandin E was analyzed using normal bone marrow cells. In primary methylcellulose culture, the addition of prostaglandin E1 (PGE1) to unseparated buffy coat, low-density, or nonadherent low-density (NAL) marrow cells resulted in an enhancement of the total number of erythroid (BFU-E) colonies observed. Treatment of bone marrow cells with a monoclonal antihuman MHC class II antibody plus complement (C') resulted in a reduction of the total number of colonies by approximately 50% and abrogation of the enhancing effect of PGE1. Analysis of accessory cell requirements by depletion of both adherent cells and sheep erythrocyte rosetting lymphocytes (E+ cells) and reconstitution using C' or anti-MHC class II antibody plus C'-treated T cell-depleted NAL (NALT-) marrow cells and E+ cell populations treated with C' or anti-MHC class II antibody plus C' demonstrated a requirement for MHC class II antigen-T cells, but not adherent cells, and a requirement for MHC class II antigen + BFU-E in order to observe the enhancing effect of PGE1 on erythroid colony formation. Positive selection of BFU-E in NALT- bone marrow expressing differing density distributions of MHC class II antigens was accomplished with monoclonal anti-MHC class II antibodies and sorting with a fluorescence-activated cell sorter (FACS). Addition of E+ cells to the different populations of MHC class II antigen+ NALT- cells demonstrated that the PGE-enhancing effects on erythroid colony formation were directly related to increasing density distributions of MHC class II antigens on BFU-E. Colony formation by BFU-E expressing a low density distribution of MHC class II antigens or having no detectable MHC class II antigens, as determined by FACS analysis, was not enhanced by PGE1 in the presence of MHC class II antigen-positive or -negative T cells.     

Microenvironmental toxicity of azidothymidine: partial sparing with hemin

Azidothymidine (AZT) is a useful drug in management of AIDS. Nevertheless, its hematologic toxicity such as anemia and neutropenia present further complications to an already compromised hematopoietic state in patients. We studied the effects of AZT on human and murine bone marrow (BM) colony growth as determined by assays of CFU-E, BFU-E, CFU-GM, and fibroblastoid stromal (CFU-Fb) colonies. Cultures were grown in methylcellulose with growth factors and scored after three- to 14-day incubation. In general, murine marrow cultures were more sensitive to AZT as compared with human marrow. Furthermore, interindividual variation in toxicity to AZT was observed between marrow samples; 1 mumol/L AZT inhibited murine CFU-E, BFU-E, and CFU-GM by 98% to 100%, whereas human marrow was inhibited by 52%, 87%, and 65%, respectively. Lower concentrations of AZT (0.1 mumol/L) inhibited murine erythroid colony growth by 85% to 90%, whereas human growth was inhibited by only 39% to 52%. Myeloid colony inhibition was similar for human and murine systems. CFU-Fb growth was markedly suppressed (75%) by 1 mumol/L AZT. Hemin, at a concentration of 10 mumol/L, overcame some of the inhibitory effects of 1 to 0.1 mumol/L AZT without hindering antiviral activity. Inhibition of human CFU-E growth was completely overcome with hemin, whereas CFU-GM growth was recovered to 66% to 74% of control. A similar but less pronounced effect was observed for BFU-E. Furthermore, hemin does not decrease AZT's effects of HIV antigen content in vitro. We conclude that anemia and neutropenia, occurring as a result of AZT, may not be as pronounced in the presence of hemin. Furthermore, CFU-Fb was significantly reduced in the presence of low concentrations of AZT. This may indicate a major target site for BM toxicity since the stromal microenvironment may be responsible for maintaining short- and long-term hematopoiesis.     

Modulation of erythropoiesis and myelopoiesis by exogenous erythropoietin in human long-term marrow cultures

In spite of their ability to support myelopoiesis for several months, human long-term marrow cultures (LTMC) are unable to sustain the production of mature erythroid cells for greater than 4 weeks. Because this preference correlates with the presence of myeloid growth factors and possible absence of erythroid factors in LTMC, we studied the effects of the erythroid growth and differentiation factor erythropoietin (Epo) on both erythropoiesis and myelopoiesis in human LTMC. Either natural or recombinant Epo was added weekly to LTMC for 10 weeks, and total cell number, numbers of hemopoietic progenitors (mixed lineage colony-forming units, CFU-MIX; erythroid burst-forming units, BFU-E; erythroid CFU, CFU-E; granulocyte-macrophage CFU (CFU-GM); granulocyte CFU, CFU-G; and macrophage CFU, CFU-M), erythroblasts (early and late), granulocytes, and macrophages were quantitated separately in the adherent and nonadherent layers of the cultures. In the absence of Epo, mature erythroid cells disappeared within the first 3-4 weeks, whereas in cultures supplemented with Epo, erythropoiesis was supported for up to 8 weeks. Results indicate that erythroid maturation is blocked at the BFU-E stage and that exogenous Epo may act on a mature subpopulation of BFU-E located in the nonadherent fraction of the cultures, promoting its maturation into CFU-E, which in turn develop into erythroblasts. However, despite Epo supplementation, erythropoiesis was not restored to in vivo proportions, suggesting that additional factors or conditions necessary for erythropoiesis are lacking in LTMC. Interestingly, we found that exogenous Epo reduced the numbers of presumably more mature (nonadherent) myeloid CFU (CFU-C), granulocytes, and macrophages compared to controls and did not alter the levels of any of the most primitive hemopoietic progenitors measured (CFU-MIX, adherent BFU-E, and adherent CFU-C). Thus the data show that exogenous Epo modulates hemopoiesis in human LTMC, enhancing erythropoiesis and suppressing myelopoiesis, but that its effects appear limited to modulating levels of the nonadherent (more mature) progenitors, leaving the numbers of the adherent (immature) progenitor cells unchanged.     

The regulatory role of interleukin 2-responsive T lymphocytes on early and mature erythroid progenitor proliferation

To analyze the role of T lymphocytes in human erythropoiesis, we evaluated the effect of recombinant interleukin 2 (IL 2) on marrow CFU- E and BFU-E colony formation in vitro. IL 2 resulted in an increase in CFU-E and BFU-E colony numbers in a dose-dependent manner. This increase could be prevented by anti-Tac, a monoclonal antibody to the IL 2 receptor. Moreover, anti-Tac on its own resulted in an overall decrease in colony numbers. Depletion of marrow adherent cells did not alter the effect of either IL 2 or anti-Tac on colony growth. Following the removal of marrow T lymphocytes, CFU-E and BFU-E colony formation proceeded normally; however, the effects of IL 2 and anti-Tac were markedly diminished. Readdition of T lymphocytes to the cultures restored the IL 2 effect. Although T lymphocytes were not themselves essential for in vitro erythropoiesis, our studies suggest that IL 2 and IL 2-responsive T cells can regulate both early and mature stages of erythroid differentiation.     

Erythropoietin receptor characteristics on primary human erythroid cells.

Erythropoietin (EP) exerts its effects on erythropoiesis by binding to a cell surface receptor. We examined EP receptor expression during normal human erythroid differentiation and maturation from the burst-forming unit-erythroid (BFU-E) to the reticulocyte level. In contrast to previous studies, we assessed EP receptor number and affinity in erythroid precursors immunologically purified from fresh bone marrow aspirates or fetal liver samples and in reticulocytes purified from peripheral blood. EP receptors were quantitated by equilibrium binding experiments with 125I EP. We found that purified primary erythroblasts from both adult and fetal sources exhibited a single high-affinity (kd 100 pmol/L) binding site for EP under our experimental conditions, and 135 or 250 receptors per cell, respectively. Reticulocytes were devoid of EP receptors. We compared these data to in vitro-derived BFU-E progeny at both early and late stages of maturation. Cultured BFU-E progeny also displayed a single class of receptors of slightly lower affinity (210 to 220 pmol/L). Preparations enriched in colony-forming units-erythroid (CFU-E) and proerythroblasts (day 9 BFU-E progeny) displayed approximately 1,100 receptors per cell, whereas populations containing mature erythroblasts (day 14 BFU-E progeny) exhibited approximately 300 receptors per cell. Furthermore, information from binding experiments was complemented by autoradiography in both enriched BFU-E preparations, cultured BFU-E progeny (days 9 and 14), and marrow mononuclear cells. These studies are consistent with a peak in EP receptor expression at the CFU-E/proerythroblast stage and a decrease with further maturation to undetectable levels at the reticulocyte stage. These data examining EP receptor characteristics on freshly isolated erythroid precursor cells complement previous data on EP receptor biology using culture-derived erythroblasts.     

Interleukin-4 suppresses the interleukin-3 dependent erythroid colony formation from normal human bone marrow cells

Human recombinant interleukin 4 (IL-4) was studied for its effects on the erythroid burst forming unit (BFU-E) from human bone marrow cells. IL-4 alone neither supports nor suppresses the erythropoietin (Epo)-dependent colony formation. Different results were obtained when IL-4 was combined with interleukin-3 (IL-3) in the presence of Epo. IL-4 suppressed the IL-3 supported erythroid colony formation in all cases (an increase of 58 +/- 8% with IL-3 versus an increase of 14 +/- 7% with IL-3 plus IL-4, n = 8). This antagonizing effect was dependent on the continuous presence of IL-4 in the culture medium, but was independent of adherent cells, B-, T-cells, or the presence of serum in the culture medium. Finally, the effects of IL-4 and IL-3 were studied on the 'Epo-independent' BFU-E by adding Epo on day 3. A decline of the IL-3 supported BFU-E was observed in the presence of IL-4 but the degree of reduction was equivalent to the results obtained when Epo was supplied at day 0. These findings indicate that IL-4 acts as suppressive growth factor for the IL-3 supported erythroid colony formation from human bone marrow cells.     

Enhancement of human erythroid progenitor cell growth by media conditioned by a human t-lymphocyte line

Recent studies have shown that soluble factors elaborated by human T lymphocytes enhance erythroid burst formation by human peripheral blood null cells. This study demonstrates that media conditioned by a long- term T lymphocyte line augmented the growth of erythroid colonies in vitro in the presence of erythropoietin (Ep). ATCC.CCl 119 (CCRF-CEM) was derived from a patient with ALL of T-lymphoblast origin. Cells from the stocks used in these experiments maintained T-cell characteristics as determined by histochemical and rosetting techniques. Increased numbers of 16 day BFU-E were seen when Ficoll-Hypaque separated peripheral blood leukocytes were cultured in the presence of a 10% (v/v) concentration of CCL 119 conditioned medium (CM). CM increased the number of BFU-E even when Ep or fetal calf serum were not growth limiting. CM also increased the number of late BFU-E observed in cultures of nonadherent bone marrow cells. When peripheral blood mononuclear cells were depleted of E-rosetting cells, only small numbers of BFU-E grew. Addition of 119 CM increased the numbers of BFU- E in E-rosette-depleted cultures. CM from B-cell, macrophage, or other T-cell lines tested did not stimulate BFU-E growth as consistently. These studies indicate that CM obtained from ATCC.CCL 119 cells contained burst-promoting activity, one of the factors required for proliferation of early erythroid progenitors.     

Effects of dexamethasone on erythroid colony and burst formation from human fetal liver and adult marrow

S ummary . Dexamethasone, a prototypic synthetic glucocorticoid, was added to cultures of human fetal liver and adult marrow cells to assess its effects on erythroid colony and burst formation. Dexamethasone decreased the number of fetal liver erythroid colonies and bursts formed in the presence of erythropoietin, and also decreased the number of cells per colony. The amount and type of haemoglobin produced per cell were unaffected by adding dexamethasone to the cultures. Dexamethasone inhibited the incorporation of ³H-thymidine into DNA in fetal liver cells stimulated with erythropoietin, supporting the hypothesis that dexamethasone inhibits the proliferation but not the differentiation of fetal liver CFU-E and BFU-E. In contrast, addition of dexamethasone to adult bone marrow cultures had a variable effect on erythroid colony and burst formation.     

Prostaglandin E acts at two levels to enhance colony formation in vitro by erythroid (BFU-E) progenitor cells

The prostaglandin E (PGE) enhancement of erythroid colony formation by human bone marrow erythroid progenitor cells (BFU-E) is mediated by a T8+ subset of lymphocytes. Medium was conditioned by bone marrow and blood T-lymphocytes and T-lymphocyte subsets (T8+, T8-, T4+, and T4- cells) in the absence or presence of PGE1 in order to determine if the cells could release a cell-free source of erythroid colony enhancing activity and what the conditions for this release would be. The T-lymphocyte conditioned medium was assayed for its effects on erythroid colony formation by nonadherent low-density T-lymphocyte depleted (NALT-) bone marrow cells plated in the presence of erythropoietin, hemin, phytohemagglutinin-stimulated leukocyte conditioned medium, or medium conditioned by 5637 cells, in the absence or presence of PGE1 and in the presence or absence of serum. PGE1 induced the release of an erythroid colony enhancing activity from the T8+ and T4-, but not from the T8- and T4+ subsets of lymphocytes, but this cell-free source of activity was only apparent if it was tested for colony formation in the presence of added PGE1. The release and action of the PGE1 induced T-lymphocyte erythroid enhancing activity did not require the presence of serum. Erythroid colony formation by NALT- bone marrow cells was not enhanced by PGE1 alone, by medium conditioned by T-lymphocytes in the absence of PGE1, or by PGE1 plus medium conditioned by T-lymphocytes in the absence of PGE1. The results suggest that the PGE1 enhancement of erythroid colony formation occurs by an apparently synergistic action on non-T-lymphocytes by PGE1 itself and by a factor or factors released from T8+ lymphocytes in response to PGE1.