Synergism between hemopoietic growth factors (HGFs) detected by their effects on cells bearing receptors for a lineage specific HGF: assay of hemopoietin-1

The preceding paper describes a new approach to the detection and assay of growth factors for developmentally early multipotent hemopoietic cells (Bartelmez et al., J. Cell. Physiol., 1985). This approach, involving measurement of the increase in the number of receptors for the mononuclear phagocyte specific hemopoietic growth factor (HGF), colony stimulating factor-1 (CSF-1), in cultures of developmentally early murine cells incubated with putative HGFs, has been used to define and assay hemopoietin-1. Hemopoietin-1 (Mr approximately 20,000) is found in the medium derived from serum-free cultures of cells of the human urinary bladder carcinoma line 5637. In contrast to both hemopoietin-2 and CSF-1, which also stimulate an increase in CSF-1 receptor numbers in cultures of developmentally early hemopoietic cells, hemopoietin-1 alone has no detectable effect. However, hemopoietin-1 exhibits dramatic synergism with CSF-1. In the presence of CSF-1, hemopoietin-1 stimulates the proliferation of developmentally earlier cells than those that respond to either CSF-1 alone or hemopoietin-2 alone or their combination. These cells proliferate for at least 3 days with no alteration of the average CSF-1 receptor density. However, by 5 days of incubation, the progeny of developmentally early hemopoietic cells that have proliferated in response to hemopoietin-1 + CSF-1 exhibit an approximately tenfold increase in the average CSF-1 receptor density per cell, which immediately precedes their differentiation to adherent mononuclear phagocytes. As hemopoietin-1 does not possess colony stimulating or burst promoting activities for murine bone marrow cells, but acts on multipotent hemopoietic cells, the analysis of the mechanism of its synergistic effects with HGFs such as CSF-1 are of special relevance to the regulation of early events in hemopoiesis.     

Hierarchical down-modulation of hemopoietic growth factor receptors

Granulocytes and macrophages can be produced in vitro when progenitor cells from mouse bone marrow are stimulated by any of four distinct colony stimulating factors, Multi-CSF (IL-3), GM-CSF, G-CSF, and M-CSF (CSF-1). At 0 degrees C the four CSFs do not cross-compete for binding to bone marrow cells, indicating that each has a specific cell surface receptor. However, at 21 degrees C or 37 degrees C, Multi-CSF inhibits binding of the other three CSFs and GM-CSF inhibits binding of G-CSF and M-CSF. Rather than competing directly for receptor binding, the binding of Multi-CSF, GM-CSF, or G-CSF to their own receptor induces the down-modulation (and thus activation) of other CSF receptors at 37 degrees C. The pattern and potency of down-modulation activity exhibited by each type of CSF parallels the pattern and potency of its biological activity. We propose a model in which the biological interactions of the four CSFs are explained by their ability to down-modulate and activate lineage-specific receptors.     

Interleukin-6 is used as a growth factor by virulent Mycobacterium avium: presence of specific receptors.

In this paper, we examined the contribution of the lymphokine interleukin-6 (IL-6) to the growth of four virulent strains of Mycobacterium avium and the nature of the binding moieties on the mycobacteria. First, we showed that human or mouse recombinant interleukin-6 are potent growth factors for four strains of virulent M. avium. This was shown to occur in tissue culture medium, which does not support maximal growth of M. avium. Bioactive IL-6 was required, inasmuch as heat-activating IL-6 or adding an antibody against IL-6 blocked this growth-enhancing ability. The rapid uptake of IL-6 by M. avium was indicated by the fact that the incubation of IL-6 with the four M. avium strains led to a rapid removal of the bioactivity from the culture medium and a rapid removal of radiolabeled IL-6. Scatchard analysis of receptor interaction showed that the M. avium strains had a single receptor species with a Kd of 50 nM and the number of receptor sites was approximately 15,000 bacterium. Blocking experiments showed that the binding of radiolabeled IL-6 was fully displaceable with cold IL-6, but not with other lymphokines. These data suggest that IL-6 may play an important role in the pathogenesis of M. avium infections, notably by promoting growth of M. avium, and that some virulent M. avium strains bind IL-6 in a specific manner.     

Proto-oncogene c-ros codes for a molecule with structural features common to those of growth factor receptors and displays tissue specific and developmentally regulated expression.

A recombinant DNA clone containing cellular sequences homologous to the transforming sequence, v-ros, of avian sarcoma virus UR2 was isolated from a chicken genomic DNA library. Heteroduplex mapping and nucleotide sequencing reveal that the v-ros sequences are distributed in nine exons ranging from 65 to 204 nucleotides on cellular ros (c-ros) DNA over a range of 11 kilobases. Comparison of the deduced amino acid sequences of c-ros and v-ros shows two differences: v-ros contains a three-amino-acid insertion within the hydrophobic domain presumed to be involved in membrane association, and (ii) the carboxyl 12 amino acids of v-ros are completely different from those of the deduced c-ros sequence. The deduced amino acid sequence of c-ros bears striking structural features similar to those of insulin and epidermal growth factor receptors, including the presumed hydrophobic membrane binding domain, amino acids flanking the domain, and the distance between the domain and the catalytic region of the kinase activity. The expression of c-ros appears to be under a very stringent control. When tissues at various stages of chicken development were analyzed, only kidney was found to contain a significant level of c-ros RNA. The level of c-ros RNA in kidney tissue is most abundant in 7- to 14-day-old chickens. Finally, nucleotide sequences of c-ros DNA and UR2-associated helper viral genome at regions corresponding to the gag ros recombination site suggest that the junction has been formed by RNA splicing.     

Sarcoma growth factor (SGF): specific binding to epidermal growth factor (EGF) membrane receptors

Cells transformed by murine sarcoma viruses (MSV) produce and release into their tissue culture media several polypeptide growth stimulating factors. One of these has been partially purified using Bio-Gel P-60 column chromatography followed by DEAE-cellulose chromatography. This growth factor was assigned the name sarcoma growth factor (SGF), and is here shown to require the epidermal growth factor (EGF) receptor in order to function as a growth factor. DEAE-cellulose chromatography yielded a product that was several-fold purer than the material present in the Bio-Gel P-60 column pool II. The biologically active material from the DEAE-cellulose column, when labeled with 125I, showed specific binding to EGF membrane receptors. The specific binding could be prevented with the addition of either unlabeled EGF or SGF. Both radiolabeled SGF and EGF will bind to live or fixed cells. We were able to bind 125I-SGF as well as 125I-EGF to fixed cells and elute the bound material from fixed receptors. The eluted SGF showed a greater than 25-fold increase in specific binding. The biological activities of EGF and SGF could be bound to and eluted from fixed receptors. The eluted SGF showed a greater than 25-fold increase in specific binding. The biological activities of EGF and SGF could be bound to and eluted from fixed cells. A 3T3 clone lacking EGF receptors was unable to respond to either EGF or SGF, whereas it responded well to serum and several other purified growth factors. The SGF isolated using DEAE-cellulose chromatography was unable to compete in a radioimmune assay using 125I-EGF and antibody to purified mouse submaxillary gland EGF; it also was not precipitated by anti-EGF antibody. From these studies it appears that the SGF produced and released by these MSV-transformed cells combines with and requires the EGF receptor in order to exert its biological effects. The peptide, however, is antigenically distinct from mouse submaxillary gland EGF.     

Purification to apparent homogeneity of a factor stimulating the growth of multiple lineages of hemopoietic cells

A glycoprotein that stimulates the proliferation of multiple hemopoietic stem and progenitor cell types was purified to apparent homogeneity. The factor, termed P cell-stimulating factor (PSF), was assayed by its ability to support the growth of murine factor-dependent hemopoietic cell lines operationally termed persisting cells (P cells). PSF was purified 50,000-fold from serum-free medium conditioned by the myelomonocytic cell line WEHI-3B by sequential ammonium sulfate precipitation, phenyl boronate chromatography, gel filtration on Sephadex G-100, neuraminidase treatment, Mono Q anion exchange chromatography, reverse phase high performance liquid chromatography on a C18 silica column, and two steps of high performance gel permeation chromatography on a TSK 3000 SW column operated under first neutral and then acidic solvent conditions. Although purified PSF could not be detected on sodium dodecyl sulfate-polyacrylamide gels stained with silver, following electrophoresis of purified PSF labeled with iodine-125, autoradiography showed only a single broad band of Mr = 30,000. This labeled band corresponded to the profile of PSF activity eluted from polyacrylamide gel slices. After reduction, labeled PSF had a slightly higher Mr of 32,000, although reduction resulted in loss of 98% of PSF activity, thus suggesting that the integrity of internal disulfide bond(s) was required for activity. When purified PSF was chromatographed on a TSK 3000 SW column under denaturing conditions in 0.1% sodium dodecyl sulfate, the single peak of absorbance at 280 nm coincided with a sharp peak of biological activity. The following unique NH2-terminal amino acid sequence of the purified PSF was obtained: NH2ALA -SER-Ile-Ser-X-X-Asp-Thr-His-Arg-Leu-Thr-Arg-. The concentration of PSF required for half-maximal stimulation of P cell growth was estimated as 1.3 X 10(-13) M or 4 pg/ml. The availability of purified PSF will allow rigorous examination of the hypothesis that a single molecule acts on multiple hemopoietic cell lineages.     

Analysis of competence: receptors for fibroblast growth factor in early Xenopus embryos

Xenopus ectodermal cells have previously been shown to respond to acidic and basic FGF by differentiating into mesodermal tissue. In the present study, ectodermal explants from Xenopus blastulae were shown to have high affinity binding sites for 125I-aFGF (Kd = 1.4 X 10(-10) M). The total number of sites, determined by Scatchard analysis, was 3 X 10(8) per explant (surface area of approximately 1 mm2). Two putative receptors of relative molecular mass 130,000 and 140,000 were identified by chemical crosslinking to 125I-aFGF. Both acidic and basic FGF, but not TGF beta 2, could compete for affinity labelling of these bands. The receptor density at the cell surface parallels the developmental competence of Xenopus animal pole cells to respond to FGF. Receptors are present at highest density in the marginal zone but are not restricted to cells in this region.     

Distribution and comparison of receptors for leukemia inhibitory factor on murine hemopoietic and hepatic cells

Leukemia inhibitory factor (LIF) is a glycoprotein that induces the differentiation of the monocytic leukemia cell line M1 but suppresses the differentiation of totipotent embryonic stem cells. In an attempt to define the normal cellular targets for LIF, the distribution of LIF receptors within hemopoietic and hepatic tissue was analyzed by binding cells with radioiodinated LIF (¹²⁵I-LIF) and subsequently carrying out autoradiography. Autoradiography demonstrated that in each he-mopoietic tissue examined cells of monocyte/macrophage lineage were the primary cell type labeled with ¹²⁵I-LIF. Moreover, both fetal and adult parenchy-mal hepatocytes displayed higher levels of labeling than either monocytes or macrophages. The number of receptors per positive cell varied from 150 for bone marrow monocytes to 2,000 for adult hepatocytes. In each case, however, binding was of high affinity, with an apparent K_D of 34–100 pM, and binding was specific, since labeling was competed for by unlabeled LIF but not a range of other structurally unrelated growth and differentiation factors. It is suggested that LIF may play a role in regulating macrophage function and hepatic acute phase protein synthesis in response to infection.     

Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells

A growth factor for vascular endothelial cells was identified in the media conditioned by bovine pituitary follicular cells and purified to homogeneity by a combination of ammonium sulfate precipitation, heparin-sepharose affinity chromatography and two reversed phase HPLC steps. The growth factor was a cationic, heat stable and relatively acid stable protein and had a molecular weight, as assessed by silver-stained SDS-PAGE gel, of approximately 45,000 under non reducing conditions and approximately 23,000 under reducing conditions. The purified growth factor had a maximal mitogenic effect on adrenal cortex-derived capillary endothelial cells at the concentration of 1-1.2 ng/ml (22-26 pM). Further characterization of the bioactivity of the growth factor reveals that it exerts mitogenic effects also on vascular endothelial cells isolated from several districts but not on adrenal cortex cells, lens epithelial cells, corneal endothelial cells, keratynocytes or BHK-21 fibroblasts, indicating that its target cells specificity is unlike that of any previously characterized growth factor. Microsequencing reveals a unique N-terminal amino acid sequence. On the basis of its apparent target cell selectivity, we propose to name this factor vascular endothelial growth factor (VEGF).     

Levels of epidermal growth factor in mice: tissues measured by a specific radioreceptor assay.

A radioreceptor assay of Epidermal Growth Factor (EGF), which uses as binder plasma membranes prepared from target tissues, instead of specific antibodies, is described. The amount of the polypeptide hormone present in the homogenate has been measured in various tissues. Submaxillary gland and parotid are confirmed to possess the highest levels of the factor. Results obtained incubating sliced tissues with or without pilocarpine, a drug which stimulates the hormone release, suggest that the tissues under investigation can be classified in two groups: a - “target tissues” (i.e. epidermis and corneal epithelium) b- “synthetizing” tissues (i.e. submaxillary gland, parotid, liver), which release the factor under pilocarpine stimulation.     

Hidden receptors for nerve growth factor in PC12 cells

The binding of nerve growth factor (NGF) to its receptors in PC12 cells was studied in two experimental conditions: (a) cell fixation with paraformaldehyde followed by permeabilization of the plasma membrane with methanol and (b) metabolic poisoning of living cells with sodium azide. Paraformaldehyde fixation of PC12 cells causes a 60-70% reduction of NGF binding capacity; the original binding capacity is restored following permeabilization with methanol. A kinetic analysis of NGF binding under these conditions reveals a single homogeneous population of receptors at variance with experiments performed in living cells where two kinetically distinct types of NGF receptors were demonstrated [Landreth, G. E. and Shooter, E. M. (1980) Proc. Natl Acad. Sci. USA, 77, 4751-4755; Schechter, A. L. and Bothwell, M. A. (1981) Cell, 24, 867-874]. Our results suggest that a proportion of the NGF receptors in PC12 cells is hidden, i.e. not available for binding to the ligand, and in a dynamic equilibrium with exposed receptors. The existence of hidden receptors is confirmed by treatment of PC12 cells with sodium azide, which causes a 50% reduction in NGF binding capacity and protection from trypsin digestion of the remaining pool of hidden receptors. The latter become exposed at the cell surface following removal of sodium azide. Our data provide an interpretation for the as yet unsatisfactorily explained data on NGF receptors.     

Assessment of nerve growth factor specific activity in tissue culture assay

1. 1. The bioassay dose-response relationship is described for the nerve growth factor (NGF). A simple method for determination of the Biological Unit (BU) is described.

2. 2. It is proposed that the potency of any nerve growth promoting protein be expressed in terms of specific activity (BU/mg). If BU is defined in terms of the amount of protein which elicits a 3⁺ response, the specific activity is a dimensionless number.

     

A serological assay for the detection of cell surface receptors of nerve growth factor

When single-cell suspensions prepared from embroyonic day 8 (E8) chick sensory ganglia are incubated with nerve growth factor (NGF), anti-NGF antiserum, and complement, an NGF-dependent cytotoxic kill of 20 (±3)% of the ganglia cells is observed. This percentage is increased by a factor of two when only the neuronal cells are tested. No kill is observed on the nonneuronal cell population representing 50% of the ganglia dissociate. When E8 sensory ganglia cells are cultured in the presence of NGF following cytotoxic kill, the large, phase-bright NGF-reponsive neurons are missing from the culture. These results indicate that the cells recognized in the cytotoxicity assay have to carry NGF-binding sites of type I, which is the one with the higher affinity of the two types of NGF-binding sites (I and II) present on sensory ganglia cells. This conclusion is further supported by the following data: (a) half maximal cytotoxicity is reached already at a concentration of NGF which is below the K_D of binding site I; (b) a washing step which removes all NGF bound to type II receptors while leaving a high percentage of type I receptors occupied has no effect on the percentage of ganglia cells killed. Using the cytotoxicity assay the presence of high-affinity binding sites of type I can be demonstrated on sensory ganglia cells from E8 chick embryos but not from E4 embryos and not on liver and heart cells from E8 embryos. Further, type I receptor-bearing cells were detectable in the brain using this assay. At E8, NGF receptors could be detected on cells of the forebrain and the tectum but not on brain stem cells. Cytotoxic kill of forebrain cells was found to be especially high at E8 and E9, and decreased by E10.     

Specific binding of radioiodinated granulocyte-macrophage colony-stimulating factor to hemopoietic cells

The hemopoietic growth factor granulocyte-macrophage colony-stimulating factor, GM-CSF, specifically controls the production of granulocytes and macrophages. This report describes the binding of biologically-active 125I-labeled murine GM-CSF to a range of hemopoietic cells. Specific binding was restricted to murine cells and neither rat nor human bone marrow cells appeared to have surface receptors for 125I-labeled GM-CSF. 125I-Labeled GM-CSF only appeared to bind specifically to cells in the myelomonocytic lineage. The binding of 125I-labeled GM-CSF to both bone marrow cells and WEHI-3B(D+) was rapid (50% maximum binding was attained within 5 min at both 20 degrees C and 37 degrees C). Unlabeled GM-CSF was the only polypeptide hormone which completely inhibited the binding of 125I-labeled GM-CSF to bone marrow cells, however, multi-CSF (also called IL-3) and G-CSF partially reduced the binding of 125I-labeled GM-CSF to bone marrow cells. Interestingly, the binding of 125I-labeled GM-CSF to a myelomonocytic cell line, WEHI-3B(D+), was inhibited by unlabeled GM-CSF but not by multi-CSF or G-CSF. Scatchard analysis of the binding of 125I-labeled GM-CSF to WEHI-3B(D+) cells, bone marrow cells and peritoneal neutrophils indicated that there were two classes of binding sites: one of high affinity (Kd1 = 20 pM) and one of low affinity (Kd2 = 0.8-1.2 nM). Multi-CSF only inhibited the binding of 125I-labeled GM-CSF to the high affinity receptor on bone marrow cells: this inhibition appeared to be a result of down regulation or modification of the GM-CSF receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of distinct receptors for two hemopoietic growth factors (granulocyte colony-stimulating factor and multipotential colony-stimulating factor) by chemical cross-linking

Granulocyte colony-stimulating factor (G-CSF) and multipotential colony-stimulating factor (multi-CSF or interleukin 3) are two members of a family of hemopoietic growth and differentiation factors. Using biologically active radioiodinated derivatives and chemical cross-linking (predominantly with the homobifunctional reagent disuccinimidyl suberate) followed by gel electrophoresis and autoradiography, receptors for these two factors have been identified. The G-CSF receptor was identified as a single subunit protein of Mr approximately 150,000 while two molecular species able to specifically cross-link to 125I-multi-CSF were identified of Mr approximately 75,000 and 60,000. For both CSFs specificity of formation of cross-linked species was demonstrated by showing that the homologous unlabeled CSF (but not other CSFs) competed for formation of the complexes with the appropriate dose-response relation, by showing that saturation occurred over the appropriate range of 125I-CSF concentration and by showing that the cellular specificity of CSF binding paralleled that for cross-linked complex formation. The formation of cross-linked complexes was dependent on the concentration and type of chemical cross-linker, especially for cross-linking of 125I-multi-CSF. Based on a number of criteria it is suggested that the two species cross-linked to 125I-multi-CSF do not represent receptors of different affinity but, rather, two noncovalently associated subunits of a receptor complex.