Comparative analysis of hematopoietic growth factors released by stromal cells from normal donors or transplanted patients.

We compared the erythroid burst-promoting activity (BPA) and colony-stimulating activity (CSA) released under serum-deprived conditions by stromal cells derived from nine normal subjects and from nine patients after bone marrow transplantation. BPA and CSA were defined according to the capacity of the conditioned media (CM) to stimulate formation of erythroid bursts and granulocyte/macrophage (GM) colonies in serum-deprived cultures of nonadherent marrow cells. Six patients (group A) failed to establish or maintain successful allografts during the study. The remaining three (group B) did not experience problems with engraftment. CM from all stromal cell cultures contained detectable levels of BPA. Preincubation of the CM with an anti-GM colony-stimulating factor (GM-CSF) monoclonal antibody (MoAb), but not with a rabbit anti-interleukin-3 (IL-3) serum, reduced BPA by an average of 94%. CM from normal and group B stromal cell cultures contained detectable CSA, and the levels correlated with the amounts of granulocyte-CSF (G-CSF) detected by a specific bioassay. G-CSF was not detectable in medium conditioned by stromal cells from transplanted patients with poor marrow function. These results indicate that CM from stromal cells from normal subjects and transplanted patients with good marrow function contain both GM-CSF and G-CSF, while CM from stromal cells from transplanted patients with poor marrow function contain detectable levels of GM-CSF only. The reduced capacity of these stromal cells to produce G-CSF is associated with a reduced capacity of the CM to sustain GM colony formation and may be associated with the inability of these patients to sustain their neutrophil counts in vivo.     

Factors that affect human hemopoiesis are produced by T-cell growth factor dependent and independent cultured T-cell leukemia-lymphoma cells

Some laboratory results and clinical situations suggest that human T cells may be important in the regulation of growth of hematopoietic cells. Since the discovery of T-cell growth factor (TCGF), systems are now available for the long-term specific in vitro propagation of mature normal or neoplastic human T cells, providing an opportunity to study the influence of T cells on hematopoiesis. Recently, 24 cell lines from patients with cutaneous T-cell lymphoma (CTCL) and T-cell acute lymphoblastic leukemia (T-ALL) were grown with TCGF and then assessed for release of humoral factors that affect hematopoiesis. Conditioned media (CM) from these cell lines were tested for erythroid burst- promoting activity (BPA) and granulocyte colony-stimulating activity (CSA). BPA was detected in CM from 3/6 cultures of T-ALL patients and 4/6 CTCL cultures. CSA was found in the CM from 6/8 cultures of T-ALL patients, 7/12 CTCL cultures, and 3/4 CTCL cell lines that become independent of exogenous TCGF for growth. The CSA from several of the neoplastic T-cell cultures stimulated high levels of eosinophil colonies, a possible source of the eosinophilia seen in these patients. The ability of continuously proliferating human T lymphocytes, which retain functional specificity and responsiveness to normal humoral regulation, to produce factors that directly or indirectly stimulate myeloid and erythroid colony formation lends further credence to the role of T lymphocytes in regulating hematopoiesis.     

A soluble activity from adherent marrow cells cooperates with IL 3 in stimulating growth of pluripotential hematopoietic precursors

Marrow cells from 5-fluorouracil (5-FU)-treated mice formed few or no mixed erythroid colonies when plated in semisolid medium with interleukin 3 (IL 3) and erythropoietin (Ep) alone. When conditioned medium (CM) from plastic-adherent marrow or thymus cells was also included, however, growth of mixed erythroid colonies was strongly stimulated. Both IL 3 and the accessory activity (AA) had to be present at the initiation of the cultures for growth to occur. AA was also produced by a cloned immortalized line (95/1.7) of fibroblastoid marrow cells that lacked macrophage-specific cell surface markers. Colony- stimulating factor-1 (CSF-1) was also released, but not granulocyte colony-stimulating activity. When 95/1.7 CM was analyzed by gel filtration, AA eluted with an apparent size of 35 kd and separated completely from the CSF-1. Biologic assays failed to detect IL 1 or IL 3 activity in 95/1.7 CM. Growth of mixed erythroid colonies from 5-FU- treated marrow is thus stimulated by adherent marrow cell-derived factors that appear distinct not only from the known CSFs including IL 3, but also from IL 1.     

Dual role of fibronectin in hematopoietic differentiation

The adhesive glycoprotein fibronectin provides anchorage for fibroblasts and hematopoietic progenitor cells in vitro. Fibronectin also demonstrates growth factor activity for fibroblasts; however, there is no available information regarding its role as a hematopoietic growth factor. To distinguish growth factor activity of fibronectin from its anchorage activity for hematopoietic progenitors, we assessed the ability of purified human plasma fibronectin to promote human bone marrow erythroid, granulocyte-macrophage (GM) and mixed granulocyte- erythroid-macrophage-megakaryocyte (GEMM) colony formation in liquid suspension, methylcellulose, and fibrin clots under serum-free conditions. Addition of fibronectin to methylcellulose cultures, or to cultures formed in fibrin clots, using fibrinogen depleted of fibronectin by preadsorption over gelatin-Sepharose and clotted with thrombin, resulted in up to a twofold enhancement of proliferation of erythroid burst-forming units (BFU-E), erythroid colony-forming units (CFU-E), and CFU-GEMM. This effect was concentration-dependent up to a fibronectin supplement of 100 micrograms/mL. By contrast, CFU-GM proliferation was not affected by the addition of fibronectin to the cultures. Fibronectin-adherent marrow cells overlaid with liquid medium formed both early and late-appearing erythroid colonies, whereas similarly cultured plastic-adherent marrow cells did not. Erythroid colony formation was observed in cultures of fibronectin-adherent marrow cells overlaid with methylcellulose but not in cultures of plastic-adherent marrow cells under the same conditions. Finally, the erythroid growth-promoting activity of fibronectin was inhibited by arginyl-glycyl-aspartyl-serine (RGDS), a tetrapeptide that competitively blocks the interaction of fibronectin with its receptor. We conclude that fibronectin plays a dual role in hematopoiesis: providing (a) anchorage for erythroid and primitive progenitors, and (b) as a proliferative stimulus for these hematopoietic cells. Both activities may be mediated by the cell adhesion domain of the molecule.     

Expression of latent hematopoietic progenitor cells in cultures of newborn and adult baboon liver

The anatomic site of hematopoiesis changes during fetal development from the yolk sac to the liver and finally to the marrow. Factors controlling this switch in the site of hematopoiesis are unknown. We assayed erythroid colony (CFU-E) and erythroid burst (BFU-E) formation in fetal, newborn, and adult baboon liver and marrow to determine the growth requirements of primate hematopoietic progenitor cells from different anatomic sites and developmental stages. We cocultured fetal, newborn, and adult liver and marrow nonadherent cells with adherent cells from these organs to assess the role adherent cells may play in determining the site of hematopoiesis. Fetal liver, fetal marrow, newborn marrow, and adult marrow cultures formed CFU-E and BFU-E colonies in vitro. In contrast, newborn and adult liver cell cultures very rarely formed colonies. However, when newborn or adult liver nonadherent cells were cocultured with marrow adherent cells, CFU-E and BFU-E colonies were detected. The colonies that formed in the newborn and adult liver cultures were derived from the liver and not from the marrow cells or peripheral blood trapped in the liver. These data suggest that in contrast to fetal liver, newborn and adult liver may not be hematopoietic organs in normal primates in vivo because of changes in the growth requirements of hematopoietic progenitor cells present in these organs.     

Identification of CD13+CD36+ cells as a common progenitor for erythroid and myeloid lineages in human bone marrow

OBJECTIVE: To identify bipotential precursor cells of erythroid and myeloid development in human bone marrow. MATERIALS AND METHODS: Cells coexpressing CD13 and CD36 (CD13+CD36+) were investigated by analyzing cell-surface marker expression during erythroid development (induced with a combination of cytokines plus erythropoietin), or myeloid development (induced with the same cocktail of cytokines plus granulocyte colony-stimulating factor of bone marrow-derived CD133 cells in liquid cultures. CD13+CD36+ subsets were also isolated on the 14(th) day of cultures and further evaluated for their hematopoietic clonogenic capacity in methylcellulose. RESULTS: Colony-forming analysis of sorted CD13+CD36+ cells of committed erythroid and myeloid lineages demonstrated that these cells were able to generate erythroid, granulocyte, and mixed erythroid-granulocyte colonies. In contrast, CD13+CD36- or CD13-CD36+ cells exclusively committed to granulocyte/monocyte or erythroid colonies, respectively, but failed to form mixed erythroid-granulocyte colonies; no colonies were detected in CD13-CD36- cells with lineage-supporting cytokines. In addition, our data confirmed that erythropoietin induced both erythroid and myeloid commitment, while granulocyte colony-stimulating factor only supported the differentiation of the myeloid lineage. CONCLUSIONS: The present data identify some CD13+CD36+ cells as bipotential precursors of erythroid and myeloid commitment in normal hematopoiesis. They provide a physiological explanation for the cell identification of myeloid and erythroid lineages observed in hematopoietic diseases. This unique fraction of CD13+CD36+ cells may be useful for further studies on regulating erythroid and myeloid differentiation during normal and malignant hematopoiesis.     

Posttransplant hematopoiesis in patients undergoing sibling allogeneic stem cell transplantation reflects that of their respective donors although with a lower functional capability

We tested the principle of whether patient long-term hematopoiesis following allogeneic stem cell transplantation (allo-SCT) reflects the characteristics of the hematopoiesis of their respective donor. For this purpose, we analyzed bone marrow (BM) hematopoiesis using long-term cultures (LTC), delta assays, and clonogeneic assays as well as CD34+ cells and their subsets by flow cytometry in a series of 37 patients undergoing allo-SCT, and we compared it to that of their respective human leukocyte antigen-matched sibling donors in a paired study performed more than 1 year after the transplant procedure. Interestingly, the main factor that influenced post-allo-SCT BM hematopoiesis in the long term was donor hematopoiesis. Nevertheless, compared to their respective donors, patients exhibited a significantly lower number of colony-forming units granulomonocytic, burst-forming units erythroid, and immature progenitors (CD34++/CD38dim/CD90+/CD133+ cells, LTC-initiating cells, and colonies generated in the delta assay). Moreover, BM stromal function was diminished in patients undergoing allo-SCT compared to their donors. In addition, the presence of chronic graft-versus-host disease under immunosuppressive treatment also conditioned an impaired hematopoietic function. In summary, our study shows that BM hematopoiesis evaluated more than 1 year after an allo-SCT mainly reproduces that of their respective donors, although with a significantly decreased in vitro activity.     

Surface membrane vesicles from mononuclear cells stimulate erythroid stem cells to proliferate in culture

In order to examine the contribution of cell surface materials to erythroid burst-promoting activity (BPA), we separated media conditioned by a variety of human cell types into pellets and supernatants by centrifugation. When added to serum-restricted cultures of nonadherent human marrow cells, pellets contained about half of the total stimulatory activity. Freeze-fracture electron microscopy of the pellets revealed the presence of unilamellar membrane vesicles ranging from 0.10 to 0.40 microM in diameter. The amount of BPA in culture increased with added vesicle concentration in a saturable fashion. Preparation of leukocyte conditioned medium (LCM) from 125I-wheat germ agglutinin labeled cells and studies comparing the glycoprotein composition of vesicles with that of leukocyte plasma membranes suggest that LCM-derived vesicles are of plasma membrane origin. Moreover, partially purified leukocyte plasma membrane preparations also contained BPA. While disruption of vesicles by freezing/thawing and hypotonic lysis did not alter BPA, heat, trypsin, or pronase treatment removed greater than 65% of BPA, implying that vesicle surface rather than intravesicular molecules express BPA. Results of BPA assays performed in two-layer clots indicated that proximity to target cells is required for vesicle BPA expression. We conclude that membrane vesicles spontaneously shed from cell surfaces may be important regulators of erythroid burst proliferation in vitro.     

Human spleen cell generation of factors stimulating human pluripotent stem cell, erythroid, and myeloid progenitor cell growth

Mitogen-stimulated murine spleen cells produce humoral substances capable of supporting murine hematopoiesis and pluripotent stem cell proliferation in vitro. Thus, we evaluated conditioned media generated by human spleen cells (SCM) in the presence or absence of mitogens for factors stimulatory for human pluripotent (CFU-GEMM), erythroid (BFU- E), and myeloid (CFU-GM) precursors. Two and one half percent to 10% SCM stimulated proliferation of all three types of precursor cells from nonadherent buoyant human marrow target cells. Mitogen-stimulated SCM augmented CFU-GM (175% to 225%), whereas CFU-GEMM and BFU-E growth was essentially unchanged. Cell separation procedures used to determine which cells provided these microenvironmental stimuli indicated that nonadherent mononuclear spleen cells provided the bulk of the CSF-GM, whereas adherent cells (95% nonspecific esterase + monocyte- macrophages) and nonadherent cells provided similar proportions of CSF- mix and erythroid burst-promoting activity (BPA). The nonadherent cells generating high levels of CSF-mix, BPA, and CSF-GM were predominantly Leu-1-negative, ie, non-T, cells. In the presence or absence of mitogens, SCM was a more potent source (1.3- to 3.8-fold) than peripheral leukocyte CM of the growth factors for the three progenitor cell types. Specific in situ cytochemical stains for analyzing morphology of myeloid colonies demonstrated that SCM stimulated the proliferation of the same types and proportions of colonies as human placental CM, suggesting that these CMs may contain similar CSF-GMs. These data show the contribution of spleen cell subsets to the generation of hematopoietic growth factors and the responsiveness of these cells to various mitogenic stimuli.     

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.     

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.     

Ultrastructural analysis of erythroid colonies in diffusion chambers in irradiated rats. Effect of cytosine arabinoside administration on donor rats.

Ultrastructural observations of erythroid colonies in diffusion chambers implanted in lethally irradiated rats are reported. The donor cells were obtained from rats treated with cytosine arabinoside (250 mg/kg body weight) 6 h prior to seeding. The marrow of donor rats contained a high proportion of stem cells committed to the erythroid line. 9 days after implantation, vast numbers of erythroid colonies developed in the diffusion chambers. The erythroid cells were observed arranged around a central macrophage resembling erythroid islands in bone marrow. Erythroid colonies most probably differentiated from committed stem cells in the inoculum. It is postulated that development of erythroid colonies in diffusion chambers is conditioned by, among other factors, the presence of a special type of macrophages. These macrophages have the same function as those of central reticular cells (CRC) in bone marrow erythroid islands.     

Effect of Cytosine Arabinoside Administration on Donor Rats

Ultrastructural observations of erythroid colonies in diffusion chambers implanted in lethally irradiated rats are reported. The donor cells were obtained from rats treated with cytosine arabinoside (250 mg/kg body weight) 6 h prior to seeding. The marrow of donor rats contained a high proportion of stem cells committed to the erythroid line. 9 days after implantation, vast numbers of erythroid colonies developed in the diffusion chambers. The erythroid cells were observed arranged around a central macrophage resembling erythroid islands in bone marrow. Erythroid colonies most probably differentiated from committed stem cells in the inoculum. It is postulated that development of erythroid colonies in diffusion chambers is conditioned by, among other factors, the presence of a special type of macrophages. These macrophages have the same function as those of central reticular cells (CRC) in bone marrow erythroid islands.     

In vitro differentiation and proliferation of human hematopoietic progenitors: the effects of interleukins 1 and 6 are indirectly mediated by production of granulocyte-macrophage colony-stimulating factor and interleukin 3

The effect of recombinant human interleukin (IL)-1 and IL-6 on the differentiation and proliferation in vitro of human granulocyte-macrophage (GM) and erythroid progenitors has been investigated in either fetal bovine serum (FBS)-supplemented or FBS-deprived cultures. Sources of progenitor cells were unfractionated bone marrow cells or marrow cells depleted of adherent and/or T cells. Each interleukin was investigated either alone or in combination with GM-colony-stimulating factor (CSF), IL-3 and erythropoietin (Epo), or granulocyte (G)-CSF. In FBS-supplemented cultures of unfractionated marrow cells, IL-1 induced optimal GM colony growth and increased by 50% the number of erythroid bursts that formed in the presence of Epo. The addition to these cultures of a neutralizing anti-GM-CSF monoclonal antibody or of an anti-IL-3 serum decreased the growth of GM colonies by 80% and 40%, respectively. Under the same conditions, IL-6 had no effect on GM colony growth but increased by 90% the number of erythroid bursts. This effect was partially (40%) neutralized by addition of anti-IL-3 serum. IL-1 and IL-6 were weak stimuli, or had no effect at all, either alone or in combination with GM-CSF and IL-3 in FBS-deprived cultures or in FBS-supplemented cultures of nonadherent or nonadherent, T-cell-depleted marrow cells. IL-1 and IL-6 had no effect, either alone or in combination with IL-3, in maintaining the number of progenitor cells in short-term liquid suspension cultures. These results indicate that the actions of IL-1 and IL-6 on hematopoiesis are mainly indirect and mediated by the production of GM-CSF and/or IL-3 by accessory cells. However, neither IL-1 nor IL-6 alone is sufficient to stimulate production of growth factor(s) by accessory cells, and at least a second stimulus, provided by FBS, is also required. These data are in agreement with a multisignal model of regulation of the expression of growth factor genes.     

In vitro stimulatory effect of substance P on hematopoiesis

The neuropeptide Substance P (SP) is widely distributed in the peripheral nervous system. Its biologic effects have been extensively studied in the immune system. However, even though the bone marrow (BM) is innervated with SP-immunoreactive fibers and some of its cells not only express SP receptors (T and B cells, endothelial cells, and macrophages) but also produce SP (macrophages, eosinophils, and endothelial cells), the effects of SP on hematopoiesis are scanty. Furthermore, SP induces the production of hematopoietic growth factors (HGFs) (interleukin-1 [IL-1], IL-6, and tumor necrosis factor alpha) from human monocytes. In this study, we have found a potent in vitro stimulatory effect of SP (10(-8) to 10(-12) mol/L) on hematopoiesis for both erythroid and granulocytic progenitors in short-term methyl- cellulose BM cultures. SP alone, in the absence of exogenous HGFs, is able to sustain hematopoiesis in vitro. This stimulatory effect of SP is: (1) mostly mediated by the adherent cells; (2) completely abrogated by two SP receptor (SP-R) antagonists; and (3) partially reduced by anti-IL-1, IL-3, IL-6, and granulocyte-macrophage colony-stimulating factor (GM-CSF). Furthermore, it appears that the stimulatory effect of SP may be mediated by IL-3 and GM-CSF because we have also found that SP induces the release of these two cytokines from BM mononuclear cells. Considering that the SP effect occurs at concentrations as low as 10(-11) mol/L, and via a specific receptor, it appears that SP may play a physiologic role in regulating hematopoiesis, at least partially through the adherent BM cells and the release of HGFs, and may place SP, a neuropeptide, in a new category of hematopoietic regulators.     

Adrenergic modulation of erythropoiesis: in vitro studies of colony-forming cells in normal and polycythaemic man

S ummary . The effect of hormonal interactions on human erythroid colony growth has been studied using the β adrenergic agonist, isoproterenol. The growth of colonies derived from both erythroid burst-forming cells (BFU-E) and erythroid colony-forming cells (CFU-E) was enhanced in the presence of isoproterenol. While isoproterenol was effective at all concentrations of erythropoietin (Ep) in cultures of marrow cells, an increase in BFU-E-derived colonies in peripheral blood cultures could be detected only at suboptimal levels of Ep concentrations. The isoproterenol effect was blocked by an agent with β ₂ receptor specificity (butoxamine), and L-isomeric configuration (L-propranolol). The stimulatory effect appeared to be mediated by a receptor different from that for Ep or for phytohaemagglutinin-stimulated leucocyte conditioned medium. Circulating BFU-E from eight patients with polycythaemia vera were also studied. Those colonies which grew in the absence of added Ep increased with isoproterenol; in cultures from normal subjects and in patients with secondary erythrocytosis, agonist stimulation was seen only in the presence of Ep. These studies provide evidence that the growth of both normal and neoplastic erythroid progenitors may be modulated by hormonal interactions.     

CCL18/PARC stimulates hematopoiesis in long-term bone marrow cultures indirectly through its effect on monocytes

Chemokines play a role in regulating hematopoietic stem cell function, including migration, proliferation, and retention. We investigated the involvement of CCL18 in the regulation of bone marrow hematopoiesis. Treatment of human long-term bone marrow cultures (LTBMCs) with CCL18 resulted in significant stimulation of hematopoiesis, as measured by the total number of hematopoietic cells and their committed progenitors produced in culture. Monocytes/macrophages, whose survival was almost doubled in the presence of CCL18 compared with controls, were the primary cells mediating this effect. Conditioned media from CCL18-treated mature monocytes fostered colony-promoting activity that increased the number of colonies formed by hematopoietic progenitor cells. Gene expression profiling of CCL18-stimulated monocytes demonstrated more than 200 differentially expressed genes, including those regulating apoptosis (caspase-8) and proliferation (IL-6, IL-15, stem cell factor [SCF]). Up-regulation of these cytokines was confirmed on the protein expression level. The contribution of SCF and IL-6 in CCL18-mediated stimulatory activity for hematopoiesis was confirmed by SCF- and IL-6-blocking antibodies that significantly inhibited the colony-promoting activity of CCL18-stimulated conditioned medium. In addition to the effect on monocytes, CCL18 facilitated the formation of the adherent layer in LTBMCs and increased the proliferation of stromal fibroblast-like cells.     

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.     

Characterization of hemopoietic activities in media conditioned by a murine marrow-derived adherent cell line, B.Ad

The hemopoietic activities present in medium conditioned by a murine bone marrow-derived adherent cell line (B.Ad) have been studied. B.Ad-conditioned medium stimulated neutrophil, neutrophil-macrophage, and macrophage colonies in agar cultures of bone marrow cells and 90% of this activity was neutralized by antimacrophage colony-stimulating factor (anti-M-CSF). The conditioned medium supported the generation and/or maintenance of spleen colony-forming units (CFU-S) in liquid cultures and synergized with multilineage colony-stimulating factor (Multi-CSF; IL-3) to stimulate colony formation by day-3 post-5-fluorouracil (FU)-treated bone marrow cells. When used as feeder layers, B.Ad cells stimulated erythroid colony-forming units (CFU-E) and markedly enhanced erythroid burst-forming units (BFU-E) stimulation more than did maximal Multi-CSF (IL-3) and Epo stimulation. No CFU-E- or BFU-E-stimulating activities were detected in medium conditioned by B.Ad cells. Similarly, B.Ad-conditioned medium was unable to stimulate Multi-CSF (IL-3) or granulocyte-macrophage (GM)-CSF-dependent cell lines. The data suggest that medium conditioned by this bone marrow-derived adherent cell line contains M-CSF and other factors not detectable as CSFs that either directly or by means of a synergistic mechanism are able to stimulate CFU-S and colony-forming cells (CFC).     

Hematopoietic growth factors in anemia of Belgrade laboratory (b/b) rats.

In this study, the extent to which growth factor production and microenvironment might be responsible for defective erythropoiesis and granulopoiesis in anemic b/b rats is investigated. Radioimmunoassay-determined serum erythropoietin (Epo) levels are high in b/b rats and closely related to degree of anemia. The low number of erythroid progenitors in b/b rats despite a high Epo level suggested that the defective erythropoiesis could be due to a low level of burst-promoting activity (BPA). A pokeweed mitogen-stimulated medium (PWM-SCM) was prepared with b/b rat spleen cells and used in normal and anemic rat bone marrow and spleen cultures to determine BPA and other growth factor levels. No erythroid burst-forming unit-derived colonies were found but granulocyte-macrophage colony-forming units were counted in significant number, suggesting that the production of growth factors that supports the growth of granulopoietic progenitors is not significantly disturbed. Because BPA is produced mainly by T-lymphocytes, the low BPA level in b/b rat PWM-SCM raised the question of the functional capacity of T-lymphocytes. Investigations showed a decrease in the proliferative activity of b/b rat spleen mitogen-activated T-lymphocytes to about 20% of controls as well as a decrease in interleukin-2 activity in b/b rat spleen cell supernatants. These results point to defective T-lymphocytes. A study of bone marrow fibroblastoid cell colonies (CFU-F) revealed significantly lower CFU-F counts in the b/b rats. This finding is indicative of a disturbed microenvironment, which could also to some extent be responsible for decreased growth factor production and depressed hematopoiesis in the b/b rat.