Biomedical subjects

S Gillis

Publications and source records attributed to S Gillis.

At least 163 records · Page 9Linked to original sources

PubMed1987

Effects of interleukin-1 on hormone release from normal rat pituitary cells in primary culture.

The present study was performed mainly to determine whether interleukin-1 (IL-1), a polypeptide produced by immunologically activated monocytes, plays a physiological role in the regulation of adrenocorticotropic hormone (ACTH) using primary monolayer cultures of rat anterior pituitary cells. Neither human IL-1 alpha nor IL-1 beta stimulated the ACTH release from normal pituitary cells in concentrations ranging from 0.01 to 10 nM. IL-1 beta caused a slight, but significant, increase in ACTH release at a concentration of 100 nM, while IL-1 alpha did not, even at the highest dose tested. IL-1 beta exhibited a synergistic action with corticotropin-releasing factor (CRF) in ACTH secretion at 10 and 100 nM of CRF, but the interaction was not striking. Both of the monokines failed to cause any change in the secretions of growth hormone, prolactin, follicle-stimulating hormone and luteinizing hormone throughout concentrations ranging from 0.01 to 100 nM. The effects of possible sex-related differences and prolonged preincubation of cultured pituitary cells in serum-free medium prior to assay incubation were also tested, providing no significantly different findings. These results suggest that the physiological significance of IL-1 as a tissue CRF is indeed questionable and should be further clarified.

Adrenocorticotropic Hormone↗

PubMed1987

Comparative effects in vivo of recombinant murine interleukin 3, natural murine colony-stimulating factor-1, and recombinant murine granulocyte-macrophage colony-stimulating factor on myelopoiesis in mice.

Purified murine colony-stimulating factors (CSF) recombinant interleukin 3 (IL-3), natural CSF-1, and recombinant granulocyte-macrophage (GM) CSF were assessed in vivo for their effects on BDF1 mouse bone marrow and spleen granulocyte-macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitor cells in untreated mice and in mice pretreated with purified iron-saturated human lactoferrin (LF). The CSF and LF preparations did not contain detectable endotoxin (less than 0.1 ng). Mice pretreated with LF were more sensitive to the effects of CSF. In mice pretreated with LF, 2,000 U IL-3 or 20,000 U CSF-1 significantly enhanced the cycling status and absolute numbers of all progenitors, whereas 20,000 U GM-CSF significantly increased the cycling status of CFU-GM and CFU-GEMM, but had no effect on cycling of BFU-E or on numbers of any of the progenitors. The effects of CSF in mice pretreated with LF were not mimicked by 0.1-100 ng E. coli lipopolysaccharide.

Animals↗

PubMed1987

Human granulocyte-macrophage progenitors and their sensitivity to cytotoxins: analysis by limiting dilution.

Limiting dilution analysis of granulocyte-macrophage progenitor cells was performed by using adherent and T cell-depleted normal human bone marrow and the recombinant human growth factors, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Estimated frequencies for progenitor cells responding to G-CSF were one in 489 for colonies scored at day 7, and one in 1,015 for day 14 colonies. For GM-CSF the frequencies were one in 1,407 (day 7) and one in 574 (day 14). The effects of tumor necrosis factor (TNF) and lymphotoxin (LT) on the frequency of progenitors responding to either G-CSF or GM-CSF was determined. Both TNF and LT inhibited the response of cells to G-CSF, and in these cultures the frequency of progenitor cells that responded to G-CSF was reduced to less than one in 100,000 cells. In contrast, the frequency of cells able to form colonies in cultures stimulated with GM-CSF was unaltered by either cytotoxin. This differential sensitivity to cytotoxins suggests that either G-CSF and GM-CSF are acting on separate granulocyte progenitor populations or that TNF and LT selectively influence the biochemical pathways associated with the activation of receptors for G-CSF.

Bone Marrow Cells↗

PubMed1987

Interactions between purified murine colony-stimulating factors (natural CSF-1, recombinant GM-CSF, and recombinant IL-3) on the in vitro proliferation of purified murine granulocyte-macrophage progenitor cells.

Purified preparations of natural CSF-1 (nCSF-1), recombinant GM-CSF (rGM-CSF), and recombinant IL-3 (rIL-3), alone and in combination, were investigated for their proliferative effects on highly enriched murine granulocyte-macrophage progenitor cells (CFU-GM). These CFU-GM had cloning efficiencies of 62%-95% in the presence of 10% (vol/vol) pokeweed mitogen-stimulated spleen cell-conditioned medium, and contained few, if any (less than or equal to 3%), contaminating morphologically recognizable monocytes or lymphocytes. The combination of low concentrations of nCSF-1 plus rIL-3, or nCSF-1 plus rGM-CSF, increased colony number greater than additively compared to the sum of colony formation with each factor alone, whereas total aggregate (colony plus cluster) number increased additively. At plateau concentrations, the previous CSF combinations increased colony number additively. Colony size was increased when nCSF-1 plus either rGM-CSF or rIL-3 were added simultaneously at either low or plateau concentrations, when compared to the size of colonies with any of the CSFs alone. Addition of rGM-CSF plus rIL-3 demonstrated no cooperative proliferative effect on either colony number or size. It is likely that these effects are mediated at the progenitor cell level and do not require accessory cell participation.

Animals↗

PubMed1987

The effects of purified recombinant murine interleukin-3 and/or purified natural murine CSF-1 in vivo on the proliferation of murine high- and low-proliferative potential colony-forming cells: demonstration of in vivo synergism.

Purified natural murine L cell (macrophage) colony-stimulating factor (nCSF-1) and purified recombinant murine interleukin-3 (rIL-3) were administered to normal or lactoferrin-pretreated mice 20 to 24 hours before sacrifice. rIL-3 and nCSF-1 administered separately increased the percentage of macrophage high-proliferative potential colony-forming cells (HPP-CFC) and low-proliferative potential colony-forming cells (LPP-CFC) in active cell cycle. Endotoxin was not detected in the samples of nCSF-1 or rIL-3 with the Limulus lysate test, and the in vitro and in vivo hematopoietic stimulatory effects of both molecules were abolished or markedly reduced by 30 minutes' treatment at 100 degrees C, which demonstrates that the effects noted in vivo were not due to endotoxin. Combinations of low concentrations of rIL-3 and nCSF-1, which by themselves were inactive, increased the percentage of HPP-CFC and LPP-CFC in active cell cycle in a synergistic fashion. No significant change in the number of HPP-CFC or LPP-CFC per femur or femoral nucleated cellularity was observed. Thus, rIL-3 and nCSF-1 can synergize to effect the proliferation of the same cell populations in vivo.

Animals↗

PubMed1987

The opposing actions in vivo on murine myelopoiesis of purified preparations of lactoferrin and the colony stimulating factors.

The actions of purified iron-saturated human lactoferrin (LF), purified preparations of human MiaPaCa colony stimulating factor-1 (CSF-1), and recombinant murine interleukin-3 (IL-3) were evaluated in vivo in mice. Studies in vitro were compared at lowered (5%), as well as at normal incubator (20%), oxygen (O2) tension because of the potentially greater physiologic relevance of in vitro studies performed at lowered O2 tension. The results demonstrate that 1) increased release of granulocyte-macrophage colony stimulating factor (GM-CSF) in vitro from pokeweed mitogen stimulated mouse spleen cells and from human mononuclear blood cells occurred at lowered O2 tension, and that human mononuclear blood leukocytes were more sensitive to the LF-induced suppression of GM-CSF release when cells were cultured at 5%, compared to 20%, O2 tension; 2) LF administered intravenously (IV) to mice pretreated with sublethal intraperitoneal dosages of Cytoxan decreased the cycling status of marrow and spleen granulocyte-macrophage (CFU-GM), erythroid (BFU-E-2 and BFU-E-1) and multipotential (CFU-GEMM) progenitor cells and the absolute numbers of these progenitors; these effects were most noticeable if care was taken to deplete endotoxin from the LF samples prior to testing LF in vivo and if the control medium was endotoxin free; 3) endotoxin-depleted LF decreased the cycling status of marrow and spleen CFU-GM, BFU-E, and CFU-GEMM and the numbers of these progenitors in the marrows of mice previously untreated with Cytoxan; these effects were most apparent when assessment of progenitor cells and their cycling rates were evaluated in vitro at lowered (5%) O2 tension; 4) purified natural human CSF-1 increased the absolute numbers of marrow CFU-GM and the cycling status of marrow CFU-GM and CFU-GEMM in mice pretreated with LF; and 5) purified recombinant murine IL-3 stimulated proliferation of day 8 and day 12 CFU-S (colony forming unit-spleen) in mice not previously treated with Cytoxan. These results substantiate the in vivo myelosuppressive effects of LF on CFU-GM and extend these effects to erythroid and multipotential progenitor cells, provide evidence that human CSF-1 has an in vivo action in mice, and confirm the studies of others showing that IL-3 stimulates the proliferation of CFU-S in vivo.

Animals↗

PubMed1987

Modulation of Friend virus infectivity in vivo by administration of purified preparations of human lactoferrin and recombinant murine interleukin-3 to mice.

Purified iron-saturated human milk lactoferrin (LF) and purified recombinant murine interleukin-3 (IL-3) were assessed in vivo for their effects on replication of spleen focus forming viruses (SFFV) in spleens of DBA/2 mice injected with the polycythemia-inducing strain of the Friend virus complex. LF and IL-3, inoculated 2 hr prior to the administration of the polycythemia-inducing strain of the Friend virus complex, respectively decreased and increased the replication of SFFV in mice as assessed by the spleen focus forming unit assay in primary and secondary DBA/2 mice. Since virus infectivity is associated with the DNA synthetic phase of the cell cycle and it has been shown elsewhere that LF decreases and IL-3 increases the percent of hematopoietic progenitor cells in S-phase in vivo, the results suggest that the opposing actions of LF and IL-3 on replication of SFFV may reflect the actions of these molecules on cycling of the target cells for SFFV.

Animals↗

PubMed1986

Purification to homogeneity of a human hematopoietic growth factor that stimulates the growth of a murine interleukin 3-dependent cell line.

A human lymphokine derived from the 5637 bladder carcinoma has been purified to homogeneity by using sequential reverse-phase high pressure liquid chromatography. A high recovery of biological activity is obtained by using this purification. The NH2-terminal amino acid sequence shows no homology to human interleukin 1 (IL 1), human IL 2, murine IL 3, or human granulocyte-macrophage colony-stimulating factor. The growth-promoting properties of the 5637-derived factor can be rapidly assayed by using the murine IL 3-dependent 32D c1-23 cell line. The amino acid sequence described is identical to that recently described for a human granulocyte colony-stimulating factor.

Animals↗

PubMed1986

Characterization of the cell surface receptor for human granulocyte/macrophage colony-stimulating factor.

125I-labeled recombinant human GM-CSF was used to identify and characterize receptors specific for this lymphokine on both a mature primary cell, human neutrophils, and on the undifferentiated promyelomonocytic leukemia cell line, HL-60. Human GM-CSF also bound to primary human monocytes and to the myelogenous leukemia cell line, KG-1, but not to any of the murine cells known to express the murine GM-CSF receptor. In addition, although some murine T lymphomas can express the GM-CSF receptor, none of the human cell lines of T cell lineage that we examined bound iodinated human GM-CSF. Binding to all cell types was specific and saturable. Equilibrium binding studies revealed that on all cell types examined, GM-CSF bound to a single class of high affinity receptor (100-500 receptors per cell) with a Ka of 10(9)-10(10)/M. More extensive characterization with neutrophils and HL-60 cells showed that in both cases, binding of GM-CSF was rapid at 37 degrees C with a slow subsequent dissociation rate that exhibited marked biphasic kinetics. Among a panel of lymphokines and growth hormones, only human GM-CSF could compete for binding of human 125I-GM-CSF to these cells. GM-CSF can not only stimulate the proliferation and differentiation of granulocyte/macrophage precursor cells, but can modulate the functional activity of mature granulocytes and macrophages as well. No significant differences in the kinetic parameters of receptor binding were seen between mature neutrophils and the undifferentiated promyelocytic leukemia cell line HL-60, indicating that maturation-specific responses to GM-CSF are not mediated by overt changes in the binding characteristics of the hormone for its receptor.

Animals↗

PubMed1986

Expression of interleukin 2, interferon-gamma, and the IL 2 receptor by human peripheral blood lymphocytes.

Human peripheral blood T lymphocytes were treated with recombinant interleukin 2, mitogens, and dexamethasone. The resulting accumulation of mRNA for interleukin 2 (IL 2), the interleukin 2 receptor (IL 2R), and interferon-gamma (IFN-gamma) was measured. IL 2 was found to regulate the levels of each of these mRNA. The expression of mRNA for IL 2, IL 2R, and IFN-gamma correlated very well with the levels of protein observed. In populations of peripheral blood T lymphocytes, the production of IL 2 and IFN-gamma were not necessarily coordinately expressed. The sequential expression of these mRNA was investigated in order to determine whether they might be independent of the action of IL 2. IFN-gamma and IL 2 mRNA showed biphasic accumulations. IL 2 mRNA accumulated very rapidly, within 60 min after mitogen stimulation and before any detectable IL 2R mRNA accumulation. Similarly, IFN-gamma mRNA accumulated rapidly, simultaneously with IL 2 mRNA. This early peak of IFN-gamma mRNA, therefore, is likely to be independent of IL 2 action. Both IL 2 and IFN-gamma mRNA then showed later peak times of accumulation. IL 2 mRNA levels peaked at 5 hr after mitogen stimulation, whereas IFN-gamma mRNA levels peaked at 20 hr. IL 2R mRNA continued to accumulate for the full 40 hr of these kinetic experiments. The later accumulations of IFN-gamma and IL 2R mRNA and the resulting expression of the corresponding proteins may therefore be dependent on the earlier production of IL 2 and its subsequent interaction with the IL 2R on the surface of such activated T cells.

Dexamethasone↗

PubMed1986

Cloning, sequence, and expression of bovine interferon-gamma.

Bovine interferon-gamma (IFN-gamma) sequences have been isolated by screening a cDNA library with a human IFN-gamma cDNA probe. The cDNA library was constructed from RNA isolated from concanavalin A-stimulated bovine lymph node cells. The open reading frame predicts that the bovine IFN-gamma precursor is composed of 166 amino acids with a predicted m.w. of 19,393. Alignment of the amino acid sequence with human IFN-gamma indicates that mature bovine IFN-gamma is composed of 143 amino acids with a predicted m.w. of 16,858. It has an amino acid homology of 63% with human IFN-gamma, and 47% with murine IFN-gamma. Biologically active bovine IFN-gamma was synthesized in an Escherichia coli expression system.

Amino Acid Sequence↗

PubMed1986

Purification to homogeneity of B cell stimulating factor. A molecule that stimulates proliferation of multiple lymphokine-dependent cell lines.

Murine B cell stimulating factor 1 (BSF-1) was purified to homogeneity from supernatants of a stimulated thymoma cell line. A protein of 18.4 kD with a unique N-terminal amino acid sequence was identified. BSF-1 had a sp act of at least 3.28 X 10(8) U/mg. In addition to its B cell-stimulatory activity, BSF-1 also stimulated the proliferation of several IL-2- and IL-3-dependent cell lines. We conclude that BSF-1 is both a growth factor and a differentiation factor. Finally, these results also suggest additional biologic properties of BSF-1 on lineages besides B lymphocytes.

Amino Acid Sequence↗

PubMed1986

Induction of macrophage tumoricidal activity by granulocyte-macrophage colony-stimulating factor.

Monocytes are a subpopulation of peripheral blood leukocytes, which when appropriately activated by the regulatory hormones of the immune system, are capable of becoming macrophages--potent effector cells for immune response to tumors and parasites. A complementary DNA for the T lymphocyte-derived lymphokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), has been cloned, and recombinant GM-CSF protein has been expressed in yeast and purified to homogeneity. This purified human recombinant GM-CSF stimulated peripheral blood monocytes in vitro to become cytotoxic for the malignant melanoma cell line A375. Another T cell-derived lymphokine, gamma-interferon (IFN-gamma), also stimulated peripheral blood monocytes to become tumoricidal against this malignant cell line. When IFN-gamma activates monocytes to become tumoricidal, additional stimulation by exogenously added lipopolysaccharide is required. No such exogenous signals were required for the activation of monocytes by GM-CSF.

Cell Line↗

PubMed1986

Characterization of the cell surface receptor for granulocyte-macrophage colony-stimulating factor.

125I-labeled recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) was used to characterize receptors specific for this lymphokine on the surface of cells of both myelomonocytic and T-cell origin. GM-CSF binding to these cells was specific and saturable. Equilibrium binding studies revealed that on all cell types examined, GM-CSF bound to a single class of high affinity receptor (1000-5000 receptors/cell) with a Ka of 10(8)-10(9) M-1. More extensive characterization with P388D1 cells showed that binding of GM-CSF was rapid at 37 degrees C with a slow subsequent dissociation rate. Among a panel of lymphokines and growth hormones, only unlabeled natural or recombinant GM-CSF were able to compete for the binding of 125I-GM-CSF to these cells. Affinity cross-linking experiments with the homobifunctional cross-linking reagents disuccinimidyl suberate, disuccinimidyl tartrate, and dithiobis(succinimidyl propionate) resulted in the identification of a receptor protein with a Mr of 130,000 on five out of the seven cell types examined. This protein was extremely sensitive to proteolysis and in the absence of protease inhibitors was degraded to a form with an approximate Mr of 70,000. A receptor protein of Mr 180,000, in addition to the Mr 70,000 protein, was found on bone marrow cells and on P815 cells. The potential tissue-specific molecular heterogeneity associated with the GM-CSF receptor may help to explain some of the diverse biological effects associated with this growth and differentiation factor.

Animals↗

PubMed1986

Characterization of the cell surface receptor for a multi-lineage colony-stimulating factor (CSF-2 alpha).

125I-Labeled colony-stimulating factor (CSF) 2 alpha (interleukin 3, multi-CSF, and mast cell growth factor) was used to characterize receptors specific for this lymphokine on the cell surface of the factor-dependent cell line FDC-P2. CSF-2 alpha binding to these cells was specific and saturable. Among a panel of lymphokines and growth factors, only unlabeled CSF-2 alpha was able to compete for the binding of 125I-labeled CSF-2 alpha to cells. Equilibrium binding studies revealed that CSF-2 alpha bound to 434 +/- 281 receptors/cell with a Ka of 8.7 +/- 3.9 X 10(9) M-1. Affinity cross-linking experiments with the homobifunctional cross-linking reagents disuccinimidyl suberate, disuccinimidyl tartrate, and dithiobis(succinimidyl propionate) produced a radiolabeled band of Mr = 97,000 on intact cells and in purified cell membranes, while an additional band of Mr = 138,000 was produced upon cross-linking to intact cells only. The relationship between these two bands is discussed. The results indicate that the receptor for CSF-2 alpha on FDC-P2 cells consists at a minimum of a subunit of Mr = 72,500.

Cell Line↗

PubMed1986

High level stable expression of human interleukin-2 receptors in mouse cells generates only low affinity interleukin-2 binding sites.

A bovine papilloma virus-derived vector was used to direct the high level expression in mouse C127 cells of three different cDNAs encoding the human interleukin-2 receptor. These were: the previously described cDNA clone isolated from the T-cell lymphoma, HUT-102; a cDNA clone isolated from mitogen-activated, normal peripheral blood T cells; and an altered version of the HUT-102 receptor in which Ser247, believed to be the site of protein kinase C-mediated phosphorylation, has been changed to an Ala residue. Fluorescence-activated cell-sorting using a monoclonal antibody directed against the human IL-2 receptor was used to derive stable lines of C127 cells expressing from 2-6 X 10(6) IL-2 binding sites per cell. However, all of these receptors bound IL-2 with low affinity.

Amino Acid Sequence↗

PubMed1986

The effects in vivo of purified preparations of murine macrophage colony stimulating factor-1, recombinant murine granulocyte-macrophage colony stimulating factor and natural and recombinant murine interleukin 3 without and with pretreatment of mice with purified iron-saturated human lactoferrin.

The influence of purified natural colony stimulating factor-1 (CSF-1), purified recombinant granulocyte-macrophage (GM)-CSF, purified recombinant interleukin 3 (IL3) and natural IL3 were assessed in mice that were untreated or pretreated with purified iron-saturated human lactoferrin (LF) in order to first suppress myelopoiesis in the mice. S1/S1d mice responded to recombinant GM-CSF and recombinant IL3 in a manner similar to the response of their +/+ littermates. These 4 factors increased the cycling status of hematopoietic progenitors in vivo. The effects were more noticeable if myelopoiesis was first decreased by LF. The effects do not appear to be due to endotoxin contamination. It cannot be discerned from these studies whether the effects are direct ones on the progenitor cells or indirect ones mediated through growth-factor releasing accessory cells. It is possible that effects can be both direct and indirect.

Animals↗

PubMed1986

Cloning, sequence, and expression of bovine interleukin 2.

Interleukin 2 (IL-2) cDNA clones have been isolated from both human and murine sources. We report here the isolation of a cDNA clone encoding bovine IL-2. This was accomplished by screening a cDNA library constructed from lectin-stimulated bovine lymph node cells, using a human IL-2 probe. Bovine IL-2 is composed of 155 amino acids and has a predicted molecular weight of 19,555. Alignment of the amino acid sequence with human IL-2 indicates that mature bovine IL-2 is composed of 135 amino acids and has a predicted molecular weight of 15,452. It has an amino acid homology of 65% with human IL-2 and 50% with murine IL-2. Bovine IL-2 is unique among IL-2 homologs in that it has a single N-linked glycosylation site. Biologically active bovine IL-2 was synthesized in an Escherichia coli expression system.

Animals↗