Erythroid burst-promoting activity of purified recombinant human GM-CSF and interleukin-3: studies with anti-GM-CSF and anti-IL-3 sera and studies in serum-free cultures

We studied the erythroid burst-promoting activity (BPA) of recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) and Interleukin-3 (IL-3) with two experimental approaches. First we studied the effects of polyclonal antisera prepared against human GM-CSF and gibbon IL-3 on colony formation from 1,000 bone marrow null cells/dish in serum-containing culture. Both GM-CSF and IL-3 independently enhanced erythroid burst formation; however, IL-3 showed more BPA activity than GM-CSF. These data are in agreement with an emerging view that the primary targets of IL-3 are primitive progenitors and that the targets of GM-CSF are intermediate progenitors, including erythroid burst-forming units (BFU-E). The proliferation of one population of BFU- E was independent of GM-CSF or IL-3. To characterize this population of BFU-E further, we developed a serum-free culture assay for the purified progenitors by incorporating insulin-like growth factor-1 (IGF-1) to the serum-free medium. The development of erythroid bursts was supported by IL-3, IGF-1, and erythropoietin (Ep) in a serum-free culture system and to a lesser extent by the combination of GM-CSF, IGF- 1, and Ep. Although the burst-promoting ability of GM-CSF and IL-3 was again demonstrated in this system, unlike serum-containing culture Ep alone did not support burst formation. These results indicate that when fetal calf serum (FCS) is present, the culture system contains BPA that is not GM-CSF or IL-3.     

Human interleukin-9 supports formation of a subpopulation of erythroid bursts that are responsive to interleukin-3.

We have investigated the biological activities of recombinant human interleukin-9 (IL-9) on enriched hematopoietic progenitors, alone or in combination with other cytokines, including Epo, G-CSF, IL-3, and GM-CSF, under serum-containing and serum-free cultures. IL-9 alone did not support colony formation. However, IL-9 plus Epo induced erythroid burst (BFU-E) formation derived from peripheral blood (PB) progenitors. Delayed addition experiments demonstrated that a part of bone marrow (BM) derived BFU-E, which seems to be immature, only responded to IL-9 and formed erythroid bursts. The burst-promoting activity (BPA) of IL-9 was confirmed using neutralizing aIL-3, aGM-CSF, and aIL-9 antisera and serum-free culture. IL-9 supported a part of BFU-E population that respond to IL-3, which was almost identical to the number of BFU-E supported by GM-CSF. IL-9 had no additive effect on erythroid and mixed colony formation supported by IL-3. In contrast, IL-9 showed an additive effect on erythroid burst formation supported by GM-CSF in serum-free culture. These data suggest that IL-9 and GM-CSF act on distinct IL-3-responsive BFU-E population. In addition, delayed addition experiment clearly demonstrated that IL-9 supports survival and the early stage of proliferation of BFU-E. These results led us to propose that IL-9 possibly acts as a BPA and selectively supports a subpopulation of early class of BFU-E that respond to IL-3.     

L-triiodothyronine augments erythropoietic growth factor release from peripheral blood and bone marrow leukocytes

To investigate cellular mechanisms involved in thyroid hormone stimulation of erythropoiesis, we studied the response of erythroid burst-forming unit (BFU-E) proliferation to L-triiodothyronine (L-T3) in a serum-free culture system. When added directly to culture, L-T3 stimulates erythroid burst formation by normal human bone marrow cells. In contrast, granulocyte-macrophage colony formation is unaffected. Enhancement of erythroid burst formation by L-T3 required the presence of nylon wool adherent and/or B-4 antigen-positive light-density marrow populations. Addition of other erythropoietic factors including platelet-derived growth factor and insulinlike growth factor II did not abrogate this apparent cellular requirement. Pulse exposure of marrow and peripheral blood mononuclear cells (greater than 95% lymphocytes) to L-T3 accelerates the release of a soluble factor that augments BFU-E proliferation into serum-free liquid culture medium. Time-course studies show that this factor appears in conditioned medium (CM) coincidentally with erythroid burst-promoting activity (BPA). Furthermore, incubation of CM with an antibody known to react with and adsorb BPA from solution removes the inducible mitogen. Biochemical analysis of CM prepared from unexposed and L-T3 pulse-exposed cells indicates that the rate of protein appearance is accelerated by L-T3 in a fashion that immediately precedes growth factor release and that several polypeptides are quantitatively increased. We conclude that unlike erythropoietin, which is mitogenic for progenitor cells directly, L-T3 enhances BFU-E proliferation indirectly by augmenting the release of soluble BPA-like molecules from accessory cells in culture.     

Inhomogeneity of the circulating BFU-E regulation in sickle cell anaemia: accessory cells properties and BFU-E growth factor response pattern

Summary. Sickle cell anaemia (SS) patients with low (<9%) HbF levels (LFSS) are characterized by an increased number of circulating BFU-E in active DNA synthesis, and release of burst promoting activity (BPA) by unstimulated low density (LD) adherent cells. In contrast, circulating BFU-E from SS patients with high (>9%) HbF levels (HFSS) are normal in number, largely in resting phase, and their LD cells do not release BPA-like activity.
We report now that in LFSS patients, adherent cell depletion decreases BFU-E growth in culture and apparent BFU-E cycling. Furthermore, addition of conditioned media (CM) from LD cells of LFSS patients restored cycling BFU-E expression in culture. Neutralization analysis with anti-GM-CSF antibody demonstrated that GM-CSF is, at least, one factor responsible for BPA activity present in this CM. Thus, GM-CSF is constitutively produced by unstimulated monocytes in LFSS patients. In contrast, HFSS patients' adherent cell depletion increases cycling of BFU-E in culture. CM from HFSS patients inhibits BFU-E expression in culture. Hence, LD adherent cells from HFSS patients may release a yet unknown inhibitor factor(s). In addition, we report a distinct response pattern in SS patients' BFU-E to growth factor (GM-CSF, IL-3): (a) LFSS patients have a BFU-E population, equally responsive to GM-CSF and IL-3; (b) HFSS patients, have a subset of BFU-E exclusively dependent on IL-3 (20–40% of the circulating BFU-E). This pattern is very similar to that of normal BFU-E.
In conclusion, BFU-E from LFSS patients represent an actively proliferating population, equally responsive to GM-CSF and IL-3, controlled by constitutively produced GM-CSF, suggesting a unique BFU-E behaviour in SS patients with low HbF levels and high haemopoietic stress. The heterogeneous regulation of BFU-E in SS disease seems to be the epiphenomenon of HbF levels, and not vice versa.     

Erythropoietin alone induces erythroid burst formation by human embryonic but not adult BFU-E in unicellular serum-free culture [see comments]

Erythroid progenitors (BFU-E) from adult human peripheral blood generate erythroid bursts in semisolid culture supplemented with at least two growth factors, ie, erythropoietin (Ep) and interleukin-3 (IL- 3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). We have analyzed the hematopoietin(s) requirement of human embryonic BFU- E, as compared to that of adult peripheral blood progenitors: This was basically evaluated in fetal calf serum-free (FCS-) methylcellulose culture of partially or highly purified progenitors treated with human recombinant hemopoietins. At a low seeding concentration (2 x 10(3) cells/dish) purified embryonic BFU-E generated erythroid bursts when treated only with Ep: Further addition of IL-3 or GM-CSF had no effect on BFU-E cloning efficiency, although the size of bursts was increased in a dose-dependent manner, particularly with IL-3. At a similar seeding concentration (ie, 10(3) cells/dish), purified adult BFU-E efficiently generated erythroid bursts in the presence of Ep and GM-CSF or IL-3, while only few small erythroid colonies were observed in the presence of Ep alone. In a final series of experiments, unicellular FCS- cultures of purified embryonic BFU-E gave rise to erythroid bursts in the presence of Ep alone. Furthermore, the cloning efficiency induced by Ep was unmodified by further addition of GM-CSF or IL-3. Unicellular FCS- cultures of highly purified adult peripheral blood progenitors generated no erythroid bursts in the presence of Ep alone. The addition of GM-CSF or IL-3 was required to generate BFU-E colonies. These studies indicate that in human embryonic life, BFU-E require only Ep for efficient erythroid burst formation, while IL-3 and GM-CSF essentially enhance the proliferation of early erythropoietic precursors.     

Three quantitative assays for human erythroid burst-promoting activity of recombinant growth factors and of omentum-conditioned medium

Human erythroid burst-promoting activity (BPA) of recombinant growth factors and crude materials, of media conditioned by omentum tissue (OMCM), and of media conditioned by the bladder carcinoma cell line (HTB9CM) was measured by three different culture methods. Using the two-stage culture method, significant activity was shown in OMCM (137%-329% of the control), HTB9CM (102%-333%), recombinant human (rh) granulocyte-macrophage colony-stimulating factor (rhGM-CSF) (179%-220%), rh interleukin 3 (rhIL-3) (232%-676%), and rh insulin-like growth factor 1 (rh IGF-1) (106%-175%), whereas there was no significant increase in the number of erythroid bursts by the same additives when the one-stage culture or the delayed erythropoietin method was employed. Linear dose-response curves were observed in the tested range of rhIL-3 and rhGM-CSF. We also observed that 1) a larger amount of rhGM-CSF was required for the optimal stimulation of erythroid burst-forming units (BFU-E) than for the optimal stimulation of granulocyte-macrophage colony-forming units (CFU-GM), and 2) even the maximum dose of rhGM-CSF increased erythroid bursts to a lesser extent than was possible by the addition of rhIL-3. The former results implies that BPA is not the major activity of GM-CSF, and the latter result, although it is not conclusive, suggests that the GM-CSF-responsive BFU-E represent only a subset population of BFU-E responsive to IL-3. The two-stage culture is a useful assay method for screening BPA in biological materials with respect to accuracy, dose responsiveness, and reproducibility.     

Purification of a membrane-derived human erythroid growth factor.

We have purified erythroid burst-promoting activity (BPA) from human lymphocyte plasma membranes by detergent extraction followed by gel-filtration, ion-exchange, and hydroxylapatite chromatography. BPA is a heat-stable integral membrane glycoprotein of Mr 28,000 by gel filtration whose activity is eluted from NaDodSO4/polyacrylamide gels as a broad band at Mr 25,000-29,000. The growth stimulator appears to be erythroid-specific, stimulating proliferation of the human erythroid burst-forming unit (BFU-E) by up to 600% of control values when tested in serum-free bone marrow culture. In contrast, it is devoid of granulocyte/macrophage colony-stimulating factor activity and has a negligible effect on the formation of human megakaryocyte and mixed hematopoietic colonies. Polyclonal anti-lymphocyte membrane IgG, which neutralizes BPA expression in culture, completely absorbs BPA from all lymphocyte-derived sources [solubilized lymphocyte plasma membranes, membrane-containing vesicles shed into lymphocyte conditioned medium (LCM) and soluble vesicle-free LCM supernatants], suggesting that soluble and membrane-derived lymphocyte BPA are antigenically related. This membrane glycoprotein may be an important mediator of proximal cellular interactions that are known to promote erythropoiesis in vitro.     

A burst-promoting activity derived from the human bone marrow stromal cell line KM-102 is identical to the granulocyte-macrophage colony-stimulating factor

Recently, several human bone marrow stromal cell lines have established and produced hematopoietic growth factors. One of these factors, a burst-promoting activity (BPA), was purified from 6 liters of serum-free conditioned medium cultured from stromal cell line KM-102, which was stimulated by phorbol myristate acetate (PMA) and calcium ionophore A23187. This stimulation induced 60 times more production of BPA than the unstimulated control culture. BPA was purified 4000-fold by sequential fractionation using ammonium sulfate precipitation, anion-exchange and lentil lectin affinity chromatographies, high performance gel filtration chromatography, and reversed phase high performance liquid chromatography. Purified BPA gave a single broad band of protein with a molecular weight of approximately 18 kd, as assessed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The concentration required for half maximal growth of early erythroid colonies was estimated as 10 pg/ml or 0.6 pM. At a higher concentration (125 pg/ml) this factor also stimulates the growth of granulocyte, macrophage, and eosinophil colonies in agar culture. The profile of amino acid composition is very similar to that of the human granulocyte-macrophage colony-stimulating factor (GM-CSF) deduced from its complementary DNA sequence. The result of amino-terminal sequence analysis strongly suggests that the purified material consists of GM-CSF and tetrapeptide-deleted GM-CSF. Moreover, antibody against GM-CSF completely neutralized the biological activities of this factor. These results indicate that the human bone marrow stromal cell line secretes GM-CSF as a burst-promoting activity and GM-CSF may play a significant role in the interaction between stem cells and stromal cells in the hematopoietic microenvironment.     

Activin A suppresses proliferation of interleukin-3-responsive granulocyte-macrophage colony-forming progenitors and stimulates proliferation and differentiation of interleukin-3-responsive erythroid burst-forming progenitors in the peripheral blood

We examined the effects of activin A on the proliferation and differentiation of immature hematopoietic progenitors prepared from peripheral blood (PB) using methylcellulose and liquid-suspension culture. In a kinetic analysis, colony formation by PB granulocyte- macrophage colony-forming unit (CFU-GM) was delayed in a dose-dependent manner by the addition of activin A only when stimulated with interleukin-3 (IL-3), but not when stimulated with granulocyte colony- stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or stem cell factor (SCF) plus G-CSF. DNA-synthesizing CFU-GM was increased by IL-3, but this effect was abolished by activin A. In contrast, PB erythroid burst-forming unit (BFU-E) was accelerated by the addition of activin A only when exposed to IL-3 plus erythropoietin (Epo), but not when exposed to Epo or Epo plus SCF. DNA- synthesizing BFU-E was increased by IL-3 and activin A, alone and additively in combination. In a mixed culture of myeloid and erythroid progenitors, activin A increased the numbers of BFU-E and CFU-Mix colonies at concentrations of 1 and 10 ng/mL and decreased the number of CFU-GM colonies in a dose-dependent manner. However, in a liquid- suspension culture of erythroid progenitors, activin A decreased total cell count and the percentage of hemoglobin-containing cells only when cells were exposed to IL-3 plus Epo. These results indicate that activin A suppresses the proliferation of IL-3-responsive CFU-GM progenitors and stimulates the proliferation and differentiation of IL- 3-responsive BFU-E progenitors, and suggest that activin A acts as a commitment factor of immature hematopoietic progenitors for erythroid differentiation.     

Interleukin-9 stimulates the proliferation of enriched human erythroid progenitor cells: additive effect with GM-CSF

Summary In the study we report here we investigated the colony-stimulating activities of interleukin-9 (IL-9). In the presence of erythropoietin, IL-9 was found to stimulate the proliferation of relatively early erythroid progenitor cells (BFU-E) from normal human bone marrow cells depleted of mononuclear phagocytes and T lymphocytes. Neutralization experiments demonstrated that the observed BFU-E-stimulating effect was not the result of intermediate production of IL-3 or GM-CSF by residual accessory cells in response to IL-9. Accordingly, the effects of IL-9 were preserved when cell suspensions were further depleted of accessory cells using CD34 enrichment of progenitor cells. Furthermore, IL-9 did not stimulate bone marrow mononuclear cells to express mRNA for IL-3, GM-CSF, EPA (erythroid-promoting activity), or IL-4, as determined by a cDNA-PCR method. IL-9 is likely to act on a subpopulation of IL-3-responsive erythroid progenitor cells that are not stimulated by GM-CSF, since plateau concentration of IL-9 and GM-CSF had additive effects on BFU-E formation, whereas a combination of IL-9 and IL-3 did not. In addition to its burst-promoting activity, IL-9 was found to have a modest stimulatory activity on myeloid progenitor cells (CFU-GM) in some experiments, suggesting that this effect may be donor related.     

Recombinant human interleukin-4 inhibits the production of granulocyte-macrophage colony stimulating factor by blood mononuclear cells

Summary. The effect of recombinant human (rh) interleukin-4 (rIL-4) on human blood BFU-E was investigated using two populations of cells: platelet-depleted low-density mononuclear cells (FH, Pl⁻ cells), as unpurified cells, and highly purified BFU-E. When FH, Pl⁻ cells were cultured with rherythropoietin (rEp), rIL-4 inhibited BFU-E growth in a dose-dependent manner. However, the addition of rIL-4 did not affect rh-interleukin-3 (rIL-3) supported BFU-E growth. Limiting dilution analysis (LDA) of FH, Pl⁻ cells showed that rIL-4 suppressed endogenous production of burst-promoting activity (BPA) by accessory cells. Highly purified BFU-E were used as target cells to measure BPA in the conditioned medium (CM) that was prepared by FH, Pl⁻ cells. When 100 purified BFU-E were cultured in 0·5 ml clots with 20% (vol/vol) of the CM, the number of BFU-E colonies was increased by the CM. The increase was significantly reduced by the addition of the CM prepared in the presence of rIL-4, but anti-IL-4 blocked the effect of rIL-4. The concentration of IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) in CM was determined by an enzyme-linked immunoadsorbent assay (ELISA). The spontaneous production of GM-CSF but not IL-3 was detected, and this was significantly decreased in the presence of rIL-4. Anti-GM-CSF but not anti-IL-3 inhibited CM supported BFU-E growth, indicating that the main BPA in the CM is GM-CSF and that rIL-4 suppresses the spontaneous production of GM-CSF by accessory cells. From these studies, we conclude that rIL-4 has a unique mechanism as a negative regulator on erythropoiesis through the inhibition of BPA production by blood mononuclear accessory cells.     

Autologous and allogeneic suppressor lymphocyte inhibition of human erythroid colony forming unit proliferation

Certain in vitro and in vivo animal studies have supported the concept that lymphocyte-derived microenvironmental factors are important in erythroid proliferation. Considerable controversy has developed regarding the applicability of these concepts to human erythroid proliferation. We, therefore, evaluated the role of lymphocytes on human erythroid colony forming unit (CFUE) proliferation in the plasma clot system. Bone marrow (BM) was obtained from normal donors and cocultured with the following cell populations: a) cultured (6 day) lymphocytes autologous or allogeneic to BM; b) cultured lymphocytes stimulated with conconavalin A (Con A), allogeneic lymphocytes or streptokinase streptodornase (SKSD). In general, unstimulated cultured lymphocytes enhanced CFUE proliferation. In contrast, lymphocytes stimulated with Con A. SKSD, or allogeneic lymphocytes suppressed lymphocyte proliferation. The suppressor cell was concentrated in the T-cell fraction obtained by sheep red blood cell rosetting followed by ficoll-Hypaque centrifugation. Both allogeneic and autologous stimulated T-cells suppressed CFUE. Moreover, supernatants from stimulated lymphocyte cultures suppressed CFUE proliferation although the cell of origin and characteristics of the suppressive factors have not been defined. These data support the concept that lymphocytes may play an important role in modulating the human BM erythroid microenvironment.     

Hematopoiesis in limiting dilution cultures: influence of cytokines on human hematopoietic progenitor cells

We have modified a limiting dilution liquid culture assay, used to quantify hematopoietic progenitor frequency, to simultaneously assess cellular proliferation and differentiation. The frequency of colonies from cord blood obtained with recombinant human interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in combination was comparable to cultures with IL-3 alone. However, IL-3 and GM-CSF in combination were synergistic for higher granulocyte proliferation than IL-3 alone, indicating that enhanced granulocyte production occurred via action of GM-CSF on progenitor cell populations already stimulated into proliferation by IL-3. Peak proliferation was evident at 4 weeks in culture, with metamyelocytes predominating; at 6 weeks, mostly neutrophils and eosinophils were present, and eosinophils were more numerous in cultures with IL-3. Increasing concentrations of erythropoietin (epo) in liquid culture with IL-3 or GM-CSF decreased absolute granulocyte yield while stimulating erythroid proliferation. The influence of epo on lineage morphology for cells plated in methylcellulose was markedly less evident by comparison, arguing against an inductive effect of epo to shift progenitor cell lineage. This liquid culture methodology may be a useful tool for preclinical screening of cytokines on human hematopoietic progenitor cells.     

Cell cycle status of erythroid (BFU-E) progenitor cells from the bone marrows of patients on a clinical trial with purified recombinant human granulocyte-macrophage colony-stimulating factor

Human Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is active in vitro as a Burst Promoting Activity (BPA) for human erythroid (BFU-E) progenitor cells. In order to evaluate the effectiveness of GM-CSF as a proliferation-inducing stimulus for BFU-E in vivo, bone marrow cells from patients on a phase I/II clinical trial with recombinant human (rh) GM-CSF, were assessed in vitro for effects on the cycling status of BFU-E. Prior to treatment, BFU-E from marrows of the majority of these patients were in a slowly cycling state. Administration of rhGM-CSF to the patients enhanced BFU-E proliferation, and cessation of treatment with rhGM-CSF resulted in BFU-E returning to a slowly cycling state. Similar results were noted for CFU-GM. This study demonstrates that rhGM-CSF has proliferation-inducing activity for BFU-E in vivo, substantiating the in vitro BPA activity previously noted for GM-CSF, although it is not possible from the present studies in vivo to determine if this effect on BFU-E is directly or indirectly mediated.     

Developmental regulation of erythropoiesis by hematopoietic growth factors: analysis on populations of BFU-E from bone marrow, peripheral blood, and fetal liver

Fetal hematopoiesis is characterized by expanding erythropoiesis to support a continuously increasing RBC mass. To explore the basis for this anabolic, nonhomeostatic erythropoiesis, the proliferative effect of recombinant hematopoietic growth factors on highly enriched hematopoietic progenitor cells from fetal and adult tissues were compared. Fetal hepatic BFU-E, unlike adult bone marrow (BM) or peripheral blood (PB) BFU-E, were capable of proliferating in response to erythropoietin in the absence of added GM colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3), and erythropoietin (Epo) directly stimulated the expansion of the fetal BFU-E pool in suspension culture. A murine monoclonal antibody (MoAb), Ep 3, was raised against enriched fetal liver progenitor cells, which detected all fetal BFU-E and which reacted with the erythropoietin-responsive, GM-CSF/IL-3-independent fraction of adult BM BFU-E and CFU-E. All adult PB BFU-E were Ep 3- but became Ep 3+ after stimulation with GM-CSF or IL-3. These data indicate that Epo plays a unique role in fetal hepatic erythropoiesis, stimulating proliferation of immature BFU-E in addition to promoting terminal differentiation of later erythroid progenitor cells. In addition, these results demonstrate a MoAb which detects all erythropoietin-responsive progenitor cells and distinguishes the BFU-E compartments in adult BM and PB.     

Selective generation of erythroid burst-promoting activity by recombinant interleukin 2-stimulated human T lymphocytes and natural killer cells

Because T lymphocytes and natural killer (NK) cells produce a variety of growth factors and interleukin 2 (IL2) modulates the activity of both, we assessed the ability of IL2 to stimulate human T cells and NK cells to produce hematopoietic growth factors detectable in clonogenic marrow culture. Human recombinant interleukin 2 (rIL2) added directly to cultures of human bone marrow that had been depleted of monocytes or depleted of both monocytes and T cells caused no significant alteration of myeloid (CFU-GM) or erythroid colony formation. Conditioned media harvested from rIL2-stimulated (greater than 100 U/mL) peripheral blood mononuclear cells, T cells, Leu-2 cells, and Leu-3 cells all had erythroid burst-promoting activity (BPA) but lacked myeloid colony- stimulating factor (GM-CSF) or CFU-GM-inhibitory activity. These T cells were IL2 receptor-negative, and the addition of anti-IL2 receptor monoclonal antibody (anti-Tac) to T cell cultures did not abrogate this IL2-stimulated BPA production. In addition, Percoll gradient-enriched, large granular lymphocytes (LGL) were separated by fluorescence- activated cell sorting into Leu-11+ (NK) cells and Leu-11- (low-density Leu-4+ T) cell fractions. rIL2 stimulated LGL, Leu-11+ and Leu-11- cells to produce BPA but not detectable GM-CSF or CFU-GM-inhibitory activity. Leu-11+ (NK) cells were Tac-negative from days 0 through 14 of culture. We conclude that rIL2 at high concentrations stimulated T cells, Leu-2 and Leu-3 cell subsets, LGL, and NK cells to produce BPA but not GM-CSF and that this stimulation may be mediated by an IL2 receptor distinct from Tac or by an epitope of the IL2 receptor not recognized by the anti-Tac antibody.     

Action of human interleukin-4 and stem cell factor on erythroid and mixed colony formation by peripheral blood-derived CD34+c-kithigh or CD34+c-kitlow cells

We studied the interaction of interleukin (IL)-4 and other burst-promoting activity (BPA) factors, such as IL-3, granulocyte/macrophage colony-stimulating factor (GM-CSF), IL-9 and stem cell factor (SCF), on erythroid burst-forming unit (BFU-E) and erythrocyte-containing mixed (CFU-Mix) colony formation in serum-free culture. IL-4 alone did not support mixed colony formation in the presence of erythropoietin (Epo). However, IL-4 showed weak but significant BPA when peripheral blood (PB)-derived CD34⁺c-kit^low cells were used as the target population. The BPA of IL-4 was much weaker than that of IL-3, which exerted the most potent activity, as previously reported. When CD34⁺c-kit^high cells were used as the target, four factors known to have BPA, IL-3, GM-CSF, IL-9 and SCF, could express BPA. In contrast, IL-4 alone failed to support erythroid burst formation. Interestingly, IL-4 showed a remarkable enhancing effect with SCF in promoting the development of erythroid burst and erythrocyte-containing mixed colonies from CD34⁺c-kit^low and CD34⁺c-kit^high cells. Delayed addition of SCF + Epo or IL-4+Epo to the cultures initiated with either IL-4 or SCF alone clearly demonstrated that SCF was a survival factor for both BFU-E and CFU-Mix progenitors. In contrast, the survival effect of IL-4 was much weaker than that of SCF, and appeared to be more important for progenitors derived from CD34⁺c-kit^low cells than for those derived from CD34⁺c-kit^high cells. It was recently reported that CD34⁺c-kit^low cells represent a more primitive population than CD34⁺c-kit^high cells. Taken together, these results suggest that IL-4 helps to recruit primitive progenitor cells in the presence of SCF.     

Evidence for direct action of human biosynthetic (recombinant) GM-CSF on erythroid progenitors in serum-free culture

The biologic activity of human biosynthetic granulocyte-monocyte colony stimulating factor (GM-CSF) was investigated in serum-free culture of erythroid progenitors derived from adult peripheral blood. The morphology of erythroid bursts and the cloning efficiency of BFU-E under serum-free conditions were similar to those observed in dishes with fetal bovine serum (FBS). For these experiments, progenitor cells were partially purified by Ficoll-Paque density centrifugation, adherence to a plastic surface, and complement-mediated cytotoxicity of Leu-1+ elements. For some studies, blastlike cells were harvested directly from 6-day-old semisolid cultures. In serum-free culture of the light-density cell fraction, biosynthetic erythropoietin (Ep) was sufficient for formation of pure and mixed erythroid colonies whereas GM-CSF was required for granulocyte-monocytic colonies. When adherent and Leu-1+ cells were removed, or when in vitro differentiated blast cells were used as a source of progenitors, neither Ep or GM-CSF alone induced colony formation. In dishes supplemented with both growth factors, erythroid bursts were detected. Although the presence of GM- CSF alone did not induce formation of any colony or clusters, BFU-E were recorded when Ep was added 8 days later, suggesting that BFU-E could be maintained. Terminal maturation of the resulting erythroid bursts was delayed by 8 days. These results provide evidence that GM- CSF acts directly on early erythroid progenitors. Furthermore, they suggest that both Ep and GM-CSF are necessary to start the differentiation process.     

Evaluation of BPA secretory ability from blood mono-nuclear cells in uremic patients after renal transplantation or rEpo treatment

Conditioned media (CM) of phyto-hemagglutinin-stimulated lymphocytes from chronic uremic patients were studied for burst promoting activity (BPA), using methylcellulose cultures with cord blood cells as a target population. Renal transplantation procedure was followed by a prompt rise of BPA, as well as the number of blood burst-forming unit erythroid (BFU-E) and hemoglobin levels, while no change in BPA and blood BFU-E number was observed in patients receiving rEpo and recovering from anemia. Thus, low BPA secretion from blood cells may have a role in the development of anemia in uremic patients.     

The role of macrophages in the regulation of erythroid colony growth in vitro.

Depletion of macrophages from murine marrow by the use of a monoclonal anti-macrophage antibody resulted in a significant increase in the number of erythroid burst forming units (BFU-E). This increase could be neutralized by the addition back to culture of macrophages or macrophage conditioned medium indicating that the suppression was mediated by soluble factors. To further characterize this effect, the addition to culture, either alone or in combination, of interleukin-1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha), and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the growth of BFU-E and the colony-forming unit granulocyte-macrophage (CFU-GM) was examined in macrophage-containing and macrophage-depleted cultures. The addition of IL-1 alpha to culture stimulated the release of both TNF alpha and GM-CSF and acted synergistically with both cytokines, resulting in a dose-dependent suppression of BFU-E and stimulation of CFU-GM growth. The increase in CFU-GM caused by the addition of IL-1 alpha was mediated by GM-CSF but not by TNF alpha as the increase was prevented by the addition of a monoclonal anti-GM-CSF antibody but not by anti-TNF alpha. When either TNF alpha or GM-CSF was neutralized by monoclonal antibodies the addition of IL-1 alpha resulted in a significant increase in BFU-E growth. The addition of GM-CSF to culture caused a dose-dependent suppression of BFU-E that was mediated by TNF alpha, as colony number was not reduced when GM-CSF and a monoclonal anti-TNF alpha antibody were simultaneously added to culture. TNF alpha-induced suppression of BFU-E only occurred in the presence of macrophages. In macrophage-depleted cultures, a dose-dependent suppression of BFU-E could be induced if subinhibitory concentrations of IL-1 alpha or GM-CSF were simultaneously added with increasing concentrations of TNF alpha. The effects of IL-1 alpha or GM-CSF and TNF alpha were markedly synergistic so that the doses required to induce suppression when added simultaneously was only 10% of that required when either were added to culture alone. Suppression of BFU-E by GM-CSF or the combined addition of GM-CSF and TNF alpha did not require IL-1 alpha because inhibition was not neutralized by the addition of anti-IL-1 alpha antibody.(ABSTRACT TRUNCATED AT 400 WORDS)