In vitro release of physically separable factors from monocytes that exert opposing effects on erythropoiesis

In order to investigate the capacity of monocytes to release erythroid burst-promoting activity (BPA), we added media conditioned by homologous monocytes to both serum-free human and serum-restricted murine marrow culture. We found that soluble, membrane vesicle-free culture medium is a potent source of the growth factor. On the other hand, monocyte membranes or exfoliated plasma membrane vesicles elaborate a factor that inhibits erythroid burst formation by up to 100%. Inhibitory activity is expressed in a dose-dependent fashion over a wide range of concentrations (0.001 to 10 micrograms/mL) tested. Experiments with antilymphocyte plasma membrane IgG, which has been shown to neutralize both soluble and membrane-bound lymphocyte-derived BPA in human marrow culture, indicate that the expression of soluble BPA by monocytes is unaffected by these antibodies. Furthermore, while antimembrane IgG is capable of absorbing BPA from LCM supernatants, these antibodies are ineffective in removing BPA from MCM supernatants, suggesting that these two soluble growth factors may be antigenically distinct. Our findings indicate that while monocytes release soluble BPA, they are also a source of membrane-associated factors that exert inhibitory effects on erythropoiesis in vitro.     

Contractile proteins participate in release of erythroid growth regulators from mononuclear cells

We have investigated the role of contractile proteins of circulating mononuclear cells in generation of membrane-associated, erythroid growth regulatory molecules. Lymphocytes and monocytes were incubated under serum-free conditions without and with cytochalasin B, cytochalasin D, or colchicine, and effects on positive and negative erythropoietic activities were determined in cell membranes and in surface membrane vesicle-rich pellets and supernatants of dialyzed medium conditioned by the cells. In serum-free cultures of human bone marrow, plasma membranes and exfoliated membrane-derived vesicles from cytochalasin-treated lymphocytes lost their capacity to support the formation of erythroid bursts, while monocyte membrane-associated inhibitory activity was abolished by preincubation with cytochalasin. In contrast, membrane-associated activities of colchicine-treated cells were unaffected. Cytochalasin-induced alterations of membrane regulatory molecules were observed in a dose-dependent fashion over a wide range of concentrations (1 to 100 micrograms/mL) tested. However, the capacity of membrane vesicle-free supernatants of medium conditioned by lymphocytes or monocytes was unaffected by cytochalasins, regardless of drug concentration used. Lysates of cytochalasin B-treated cells inhibited the activity of deoxyribonuclease I to a greater degree than did lysates of untreated cells, suggesting that the relative amount of monomeric actin is increased in the cytoplasm of treated cells. Furthermore, results of experiments with D-glucose and with cytochalasin D suggest that cytochalasin effects are independent of alterations in glucose metabolism. The data indicate that expression of plasma membrane- associated regulators is sensitive to agents that block polymerization of actin. They raise the possibility that changes in distribution of actin between unpolymerized and filamentous pools may influence the organization and/or function of mononuclear cell surface-associated erythroid regulatory molecules.     

B-lymphocytes as a source of cell surface growth-promoting factors for hematopoietic progenitors

Although B cells reside in the bone marrow, little is known concerning their functional role in hematopoiesis. We have measured the effects of surface membrane factors released from unstimulated, circulating B cells of normal donors and patients with chronic lymphocytic leukemia on human hematopoiesis in vitro. Leukemic cells augment erythroid burst formation by allogeneic blood cells (p less than 0.05). The stimulatory effect is increased in cultures containing a high B-cell seeding density, and is decreased in those with a high peripheral blood mononuclear cell seeding density. Medium conditioned by B cells (CM) from the circulation or bone marrow stimulates the formation of erythroid bursts, granulocyte-macrophage colonies, and mixed colonies containing granulocytes, erythroblasts, monocytes, and megakaryocytes (GEMM) in serum-free cultures of allogeneic and autologous marrow (p less than 0.05). This effect is localized primarily to surface membrane vesicle-rich pellets of CM. Screening of several hematopoietic and nonhematopoietic cell types reveals that membrane vesicle-associated activity is released from B cells and mitogen-stimulated, circulating T cells. In contrast, vesicles shed from freshly isolated, resting T cells, continuous T-cell leukemia cell lines, erythrocytes, and endothelial cells do not express the activity (p greater than 0.10). The stimulatory activity is augmented in cultures of marrow cells that are depleted of B4 antigen-positive lymphocytes but not of T-lymphocytes, suggesting that endogenous release of the factor(s) occurs during incubation. Furthermore, membranes partially purified from leukemic B cells also express the activity. Together with our findings that (1) the growth enhancing factor(s) is solubilized by octylglucoside, and that (2) the factor can be immunoprecipitated with BPA-neutralizing, antimembrane IgG, our results suggest that the erythropoietic activity is an integral membrane protein that may be immunologically related to BPA. The relationship of the erythroid burst stimulatory factor to other hematopoietic activities found in CM pellets is unknown.     

Neutralization of erythroid burst-promoting activity in vitro with antimembrane antibodies

To investigate the relatedness of soluble and pelletable vesicular erythroid burst-promoting activity (BPA) present in lymphocyte- conditioned medium (LCM), we immunized rabbits with partially purified lymphocyte plasma membranes and tested the antisera for biological and immunologic crossreactivity with LCM and its component fractions. When preincubated with IgG purified from post-immune but not from preimmune serum, BPA expression by unseparated LCM, LCM-derived pellets, and supernatants was abolished in a dose-related fashion. As little as 0.001 mg/mL post-immune IgG reduced burst formation by 50%. Antimembrane IgG crossreacted on immunoblots with multiple components of both supernatants and pellets of LCM. Crossreactivity was also seen in LCM-derived supernatants that were subjected to ultracentrifugation. Soluble BPA was adsorbed from LCM supernatants incubated with antimembrane IgG-coated Staphylococcus aureus. Conversely, incubation of purified antimembrane IgG with intact circulating lymphocytes removed BPA-neutralizing effects from the antibody preparation. Antimembrane IgG incompletely suppressed erythroid colony-forming unit (CFU-E)-derived colony formation, an effect that could not be explained by alteration in erythropoietin sensitivity or action. There was no effect of the antibody preparation on erythroid differentiation of K562 cells or on CFU granulocyte/macrophage-derived colony growth, (CFU-G/M) by human or murine bone marrow. Taken together, our findings suggest that antibodies directed against lymphocyte plasma membranes react with both soluble and vesicular BPA, and that these physically separable erythroid growth factors may share antigenic determinants.     

Human macrophage colony-stimulating factor is expressed at and shed from the cell surface

Surface membrane-associated growth factors are being recognized as important for developmental processes, including cell assembly, differentiation, and growth. To investigate the role of membrane-bound macrophage colony-stimulating factor (M-CSF) in myelopoiesis, and whether this factor is released from the cell surface in association with shed membrane-derived vesicles, COS-1 cells were transfected with cDNAs for M-CSF-tau (containing the transmembrane domain) or a soluble mutant form of the molecule lacking the transmembrane domain ([s]M-CSF- alpha). COS-1 cells transfected with either cDNA released activity into the spent culture medium. Conditioned medium was separated by centrifugation into supernatants and pellets were found to contain plasma membrane-derived vesicles by transmission electron microscopy. When medium fractions were assayed in marrow cultures, activity was localized to shed plasma membrane-derived vesicles in medium conditioned by cells transfected with cDNA for M-CSF-tau and in the vesicle-free supernatants of medium conditioned by cells transfected with cDNA for [s]M-CSF-alpha. In addition, nuclear, mitochondrial, and plasma membrane subfractions of stably transfected cells were prepared and assayed for activity. Concentration-dependent stimulation of macrophage colony formation was observed with purified plasma membranes (but not nuclear or cytosolic proteins) from cells transfected with cDNA for M-CSF-tau. By contrast, membranes from untransfected cells and cells transfected with cDNA for [s]M-CSF-alpha or control DNA expressed no activity. Together, the data indicate that human M-CSF is expressed at the cell surface and exfoliated in association with surface membrane- derived vesicles.     

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 monoclonal antibody to exfoliated surface vesicles that recognizes a membrane-associated erythroid burst-promoting activity

A monoclonal antibody (MoAb) recognizing a membrane-associated erythroid burst-promoting factor was prepared by immunizing BALB/c mice with plasma membrane-derived vesicles exfoliated from lymphocytes under serum-free conditions. Hybrids secreting antibody reactive with lymphocyte plasma membranes were formally cloned and IgG was purified from monoclonal supernatants or from BALB/c mouse ascites fluid. Two clones (D3-E4 and D3-G9) were found to suppress burst forming unit- erythroid (BFU-E) proliferation when added directly to serum-free human marrow culture. Inhibition to a level of 100% was observed in a dose- dependent fashion over a wide range of antibody concentrations (0-200 micrograms/mL). Neither antibody altered the proliferation of colony forming unit-granulocyte macrophage (CFU-GM) or colony forming unit- granulocyte-erythroid-monocyte-megakaryocyte (CFU-GEMM) progenitor cells in human bone marrow culture. The D3-E4 clone was found to produce an IgG1 antibody which adsorbs an erythroid burst-promoting activity (BPA) from supernatants of, and octylglucoside extracts of shed vesicles present in, serum-free, lymphocyte conditioned medium (LCM), and which recognizes a vesicular protein of Mr approximate 30,000 on immunoblots of membrane proteins electrophoresed on sodium dodecyl sulfate (SDS)/polyacrylamide and transferred to nitrocellulose. In contrast, the D3-G9 clone was found to produce an IgG1 cytotoxic antibody. These antibodies will be important to the study of cell-cell and growth factor-cell interactions in vitro.     

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.     

Erythroid burst-promoting activity produced by interleukin-1-stimulated endothelial cells is granulocyte-macrophage colony-stimulating factor

Interleukin-1 (IL-1) induces cultured human umbilical vein endothelial cells to elaborate heterogeneous hematopoietic growth factors, including granulocyte-macrophage and granulocyte colony-stimulating factors (GM-CSF and G-CSF, respectively). Because erythroid burst- promoting activity (BPA) is also elaborated by endothelial cells exposed to IL-1, we sought to determine whether the BPA released by IL- 1-induced endothelial cells simply reflects the known erythropoietic activity of GM-CSF or whether other uncharacterized factors might be involved. Media conditioned by multiply passaged endothelial cells cultured for three days with recombinant IL-1 alpha (ECMIL-1) stimulated erythroid burst and GM colony formation in cultures of human nonadherent T-lymphocyte-depleted marrow mononuclear cells. Pretreatment with an anti-GM-CSF antiserum neutralized all the BPA and 56% of the GM colony-stimulating activity (GM-CSA) in ECMIL-1. The antiserum used in these studies did not inhibit IL-3 or G-CSF activity and did not inhibit ECMIL-1-induced murine GM colony growth (a measure of human G-CSF). To examine whether GM-CSF induces BPA release by accessory cells, media conditioned by marrow cells cultured for three days with GM-CSF were tested in the colony growth assays. Pretreatment with anti-GM-CSF antiserum completely neutralized the BPA and GM-CSA of the marrow cell-conditioned medium. We conclude that GM-CSF is the BPA elaborated by IL-1-induced endothelial cells. The in vitro erythropoietic activity of GM-CSF is not dependent on induced BPA release by accessory cells and therefore likely results from a direct effect of GM-CSF on progenitor cells.     

Stimulatory effects of plasma from patients with acute nonlymphoblastic leukemia on early erythroid progenitors and pluripotent stem cells

To examine mechanisms of cytopenia in acute nonlymphoblastic leukemia (ANLL), we determined whether leukemic plasma (LP) contains growth-promoting factors that support mammalian erythroid progenitor and pluripotential stem cell proliferation in vitro. When added to serum-free cultures of human bone marrow and peripheral blood cells, LP from anemic patients with ANLL stimulated erythroid burst formation to greater levels than did normal human plasma (p less than 0.05 for each). While LP also enhanced erythroid burst development in murine bone marrow cells, preincubation of marrow cells with LP did not alter the formation of splenic colonies (CFU-S-derived colonies) in irradiated mice (p greater than 0.10). To determine whether erythropoietin or other growth factors (functionally similar to burst-promoting activity, BPA) are important in mediating the erythropoietic effects observed in vitro, LP was preabsorbed with monospecific IgG raised against human erythropoietin or human BPA. Although elevated erythropoietin levels were found in each LP, preabsorption with antierythropoietin IgG did not alter its capacity to enhance human burst formation. In contrast, preabsorption with antimembrane IgG capable of recognizing human BPA abrogated the stimulatory effects of LP (p less than 0.05). In addition, LP was found to increase the percentage of murine CFU-S that are synthesizing DNA by the (3H) Tdr suicide technique, an effect which was not abrogated by preabsorption of LP with monospecific IgG raised against human BPA. We conclude that both erythropoietin and BPA are appropriately increased in ANLL. In addition, a factor is present in LP which induces DNA replication in murine pluripotential stem cells.     

Evidence for proliferation of erythroid progenitor cells in the absence of added erythropoietin.

The cellular mechanisms underlying the requirement of leukocyte conditioned medium for human erythroid colony formation has been studied. Using photographic methods it was found that erythroid progenitor cells were able to form small colonies in the absence of added erythropoietin (epo). The addition of epo at this time leads to further growth and hemoglobinization of cells within the colony. Leukocyte conditioned medium was found to be required for this early proliferative stage of erythroid colony formation.     

Use of lectins and polyethylene glycol for fusion of glycolipid-containing liposomes with eukaryotic cells.

Efficient fusion of phospholipid vesicles with monolayer cultures of eukaryotic cells was accomplished by attaching glycolipid-containing vesicles to the cell surface by using a lectin displaying binding for both the cell surface and the glycolipid, followed by treatment with polyethylene glycol. Fusion was inferred from the transfer of fluorescent lipid analog probes embedded in the vesicle membrane over the entire cell surface and of fluoresceinated proteins from the aqueous space of the vesicle to the cytoplasm of the cell. Fluorescence recovery after photobleaching showed that both the injected membrane and the cytoplasmic markers were mobile. Two different lectin--glycolipid combinations [Ricinus communis agglutinin I-lactosylcerebroside and concanavalin A-tetradecyl- (or hexadecyl-) maltobionamide] were used to promote attachment of lipid vesicles before polyethylene glycol-induced fusion with BG-9 human fibroblasts, NIL-8M2 hamster cells, or L-929 mouse cells. In the absence of lectin or polyethylene glycol, fusion was negligible. However, when both the lectin and the glycol were used, a dramatic increase in the transfer of both vesicle membrane and aqueous space markers from the liposomes to the cells occurred.     

Expression of stem cell factor and c-kit mRNA in cultured endothelial cells, monocytes and cloned human bone marrow stromal cells (CFU-RF).

Previously, we have shown that conditioned medium from a subpopulation of human marrow stromal cells (CFU-RF) contain an activity able to stimulate the growth of macroscopic epo-dependent erythroid colonies. The ligand for the product of the c-kit proto-oncogene (also known as stem cell factor or SCF), among other activities, has been reported to have similar effects on erythroid colony growth. We have also presented data showing that SCF together with phytohemagglutinin-stimulated leukocyte conditioned medium can stimulate erythroid colony growth in the presence of antibodies to erythropoietin. Using the human SCF cDNA probe (K. Zsebo, Amgen Inc.) we now show that cells derived from CFU-RF colonies express SCF but not c-kit. Human umbilical vein endothelial cells were also found to express SCF and this expression was increased by addition of monocyte supernatant, IL-1 beta or thrombin. Cells of the human erythroleukemia cell line HEL were found to express c-kit but not SCF. Neither c-kit nor SCF mRNA were detected in phytohemagglutinin-stimulated lymphocytes. Together, these data support the view that the behaviour of proliferating erythroid stem cells in the marrow, which may express c-kit, could be regulated by membrane-bound SCF present on surrounding stromal cells.     

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.     

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.     

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.     

Promotion of erythropoietic bursts in culture of human bone marrow by blood-derived mononuclear cells

Summary In this report we give evidence that the addition of different amounts of irradiated mononuclear cells (MNC) from normal blood to bone marrow cell cultures containing erythropoietin produces an increase in number and size of erythropoietic bursts proportional to the number of irradiated MNC added. There was no consistent difference in the use either of autologous or of allogeneic MNC as burst-promoting activity source in normal individuals. Plating efficiency studies demonstrated that the number of BFU-E detected had a linear relationship to the number of bone marrow cells plated when 5×10⁵ blood MNC were present as BPA source. A comparison between different preparations of human leukocyte conditioned media and 5×10⁵ irradiated MNC, demonstrated that in all experiments irradiated cells stimulated the growth of a higher number of marrow-derived BFU-E. Therefore the use of 5×10⁵ irradiated MNC from normal blood is proposed as standard BPA source in human bone marrow erythropoietic cultures.Research work supported by the Deutsche Forschungsgemeinschaft, the Commission of the European Communities (Euratom), The Federal Ministry of Interiors and the Volkswagen Foundation     

Macromolecules stimulating human granulocytic colony-forming cells, precursors of these cells, and primitive erythroid progenitors: some apparent nonidentities

The relationship between molecules having granulocyte colony- stimulating activity (G-CSA), erythroid burst-promoting activity (E- BPA), and activity promoting increase in the number of granulocytic progenitors in liquid culture (delta GPA) was explored in conditioned medium from human leukocytes (HLCM) and human placenta (HPCM). As tested on human hemopoietic progenitors in culture, G-CSA eluted from Sephadex G100 as a single peak with apparent molecular weight of 25,000, separating partially from E-BPA and delta GPA, which both had an apparent molecular weight of 45,000. All three activities eluted together from hydroxyapatite at low molarity phosphate. Their charge properties were also similar and all three electrofocused in flat gel beds in the pH range near 5.4. On both hydroxyapatite and isoelectric focusing, delta GPA sometimes separated partially from the other two activities but not consistently. The gel filtration result shows that in conditioned medium of human origin, molecules having G-CSA are not the same as those having delta GPA, suggesting a dual factor requirement in the granulocytic lineage reminiscent of that in the erythroid pathway. The results suggesting that delta GPA might differ from E-BPA, on the other hand, were not consistent enough to establish their nonidentity. Single micromanipulated cells proved capable of forming erythroid or granulocytic colonies in the presence of either crude or partially purified activity. The results establish that human colony-forming cells are direct primary targets of growth factors in HLCM and HPCM.     

Arrest and metastasis of blood-borne tumor cells are modified by fusion of plasma membrane vesicles from highly metastatic cells.

B16 mouse melanoma sublines in culture spontaneously shed intact plasma membrane vesicles. These vesicles can be fused with the plasma membrane of cells from homologous and heterologous B16 sublines by using polyethylene glycol and phytohemagglutinin-P. Fusion of vesicles from a highly metastatic subline (F10) that localizes exclusively in the lung with cells from a poorly metastatic subline (F1) significantly increased the ability of F1 cells to become arrested in the lung and form metastases in this organ. In contrast, fusion of F1 vesicles with F10 cells did not alter the ability of vesicle-modified cells to localize in the lung or form lung metastases. F10 vesicle-modified F1 cells reverted to their original arrest behavior and metastatic capacity after removal of F10 vesicle components from the plasma membrane. The changes in the arrest and metastatic behavior of F10 vesicle-modified F1 cells were highly highly specific. Vesicles from other B16 sublines that are poorly metastatic and show limited localization in the lung (F1, FLLr, and F10Lr) did not modify the arrest behavior and metastatic capacity of FU cells. These results suggest that the differences in the abilities of the F1 and F10 sublines to localize in the lung are determined by differences in cell surface properties.     

Internalization and cellular processing of somatostatin in primary culture of rat anterior pituitary cells

Somatostatin (SRIF) binding, internalization, and intracellular processing in primary culture of anterior pituitary cells have been studied using somatostatin coupled to an electron-opaque marker, colloidal gold. Initially, after 2 min of incubation (37 C), gold-conjugated SRIF is localized on the cell surface, with 38% of the marker being found around microvilli, 10% at the junction of secretion vesicles with the plasma membrane, and 51% distributed over the remaining areas of the cell membrane. There was no internalization of SRIF at this time. After 20 min of incubation, distribution of the cell-surface bound hormone was similar to that at 2 min (40.6% at microvilli, 12% at the junction with the secretion vesicle, and 47.4% over the rest of the plasma membrane). However, 12% of the electron-opaque markers were found intracellularly in association with coated vesicles, intermediate-sized vesicles, lysosomes, and Golgi structures. SRIF did not enter pituitary cells at 4 C. To study the role of coated vesicles in internalization of SRIF, we have measured somatostatin binding to isolated coated vesicles before and after various treatments and sonication. SRIF binding to sonicated vesicles (3.46 +/- 0.36 fmol/micrograms protein), was much greater than to intact ones (0.75 +/- 0.16 fmol/micrograms protein), suggesting intraluminal localization of SRIF receptors in the coated vesicles. Approximately 80% of SRIF-binding sites were recovered on the intraluminal surface of the coated vesicles. The results of these experiments suggest that internalization of SRIF is a time- and temperature-dependent process. Within the cell, SRIF is routed to either lysosomes or the Golgi apparatus. Coated vesicles participate in intracellular translocation of SRIF-receptor complexes. It appears that the receptor for SRIF being internalized is located on the intraluminal surface of the coated vesicle.