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)     

Selective induction of enhanced complement receptor 3 activity on murine macrophages

A high proportion of murine resident peritoneal macrophages bear complement receptors 1 and 3 (CR1, CR3) which bind C3b and iC3b components of complement, respectively. By contrast, macrophages derived from bone marrow, blood, and the elicited peritoneal exudate are predominantly CR1+3. To determine if the microenvironment of the normal peritoneal cavity influences CR3 phenotype, we studied the effects of lavage from the cavity on cultures of primary peritoneal exudate macrophages, and on macrophages derived from progenitors in the bone marrow, blood, and peritoneal exudate. The cell-free peritoneal lavage (CFPL), after 24 hr of culture, induced CR3 on primary and culture-derived populations of peritoneal exudate macrophages but had no effect on the CR3 phenotype of macrophages derived from bone marrow or blood. The CR3-inducing activity in CFPL was abolished by heating at 70 degrees C for 30 min and by trypsin, and was not affected by adsorption with EA(IgM)iC3b indicator cells, demonstrating that it is not soluble CR3. Finally, exudate macrophages exposed to CFPL required at least 24 hr before they expressed CR3; such macrophages regenerated CR3 after the receptors were removed by trypsin. The selective effect of the activity in CFPL for peritoneal exudate macrophages indicates that the local microenvironment of the peritoneal cavity can influence the expression of CR3.     

The effect of colony stimulating factor on the synthesis of ribonucleic acid by mouse bone marrow cells in vitro.

The effect of granulocyte-macrophage colony stimulating factor (GM-CSF) on the synthesis of RNA in liquid cultures of mouse bone marrow, spleen, thymus, peritoneal, peripheral blood leukocytes and lymph node cells was investigated. GM-CSF appeared to stimulate RNA-synthesis in syngeneic bone marrow cells within ten minutes of adding it to the culture. In the presence of GM-CSF bone marrow cultures maintained their initial rate of RNA synthesis for approximately ten hours. GM-CSF had no apparent effect on the uptake of 3H-uridine into bone marrow cells. This stimulation was still observed in the presence of puromycin and cycloheximide, but was abrogated by actinomycin D. The magnitude of the stimulation was not affected by the density of cells between 1 and 20 x 10(6) cells/ml but was slightly smaller at 0.1 and 40 x 10(6) cells/ml. Increasing concentration of GM-CSF (up to 2 X 105 units per ml) led to increased stimulation of RNA synthesis in bone marrow cells, but a significant stimulation could be detected at concentrations as low as 800 units/ml. GM-CSF did not significantly stimulate RNA synthesis in spleen, thymus, mesenteric or subcutaneous lymph node cells. However a small stimulation was observed in peripheral blood leukocytes and peritoneal cells. Autoradiographic studies showed that GM-CSF stimulated RNA synthesis in blast cells, myelocytes, metamyelocytes and polymorphs. Nucleated erythroid cells showed no increased labeling with GM-CFS. Labeling in lymphoid-like cells was highly variable but the level of labeling did not appear to be influenced by GM-CSF.     

A novel rat monoclonal antibody directed against a population of activated mouse peritoneal macrophages

A hybridoma clone secreting rat monoclonal antibody (MAB) designated as 3F3.5F and which reacted with a population of activated tumoricidal mouse peritoneal macrophage (M phi) was produced by the fusion of mouse myeloma cells with rat spleen cells immunized against adherent BCG-activated mouse peritoneal exudate cells (adherent BCG-PEC). The antibody was cytotoxic and of the rat IgM class. The specific reactivity of the antibody with mouse primary cells and cell lines was examined by complement-dependent cytotoxicity and indirect immunofluorescence flow cytometry analysis. The antibody was found to bind to about 40% of the adherent BCG-PEC activated in vivo and elicited peritoneal macrophages activated in vitro by lymphokine and lipopolysaccharide (LPS), to about 35% of polymorphonuclear neutrophils (PMN) 15 hr after intraperitoneal injection of BCG, to about 30% of bone marrow cells from BCG-infected mice, to about 10% of P815 mastocytoma cells and to thioglycollate-induced PEC to some degree. It did not bind to other cells tested including BCG-induced peritoneal lymphocytes, non-tumoricidal PEC, thymocytes, spleen cells, resting bone marrow cells from normal mice, lymphomas, myelomas, fibroblasts, or macrophage-cell lines. Pretreatment of adherent BCG-PEC with MAB 3F3.5F and rabbit complement caused a considerable decrease in tumor cytotoxicity toward P815 cells, but the same pretreatment of non-adherent BCG-PEC had no inhibitory effect on natural killer activity for YAC-1 cells.     

The role of tumor-derived cytokines on the immune system of mice bearing a mammary adenocarcinoma. I. Induction of regulatory macrophages in normal mice by the in vivo administration of rGM-CSF

Using a dimethylbenzanthracene-induced immunogenic nonmetastatic murine mammary adenocarcinoma in BALB/c mice, our previous work has shown that splenocytes from tumor bearers have reduced responses to both mitogens and Ag including tumor-associated Ag. NK and cytotoxic T cell activities are also reduced in splenocytes of tumor bearers. Mac-1+2+ macrophages induced in mammary tumor bearers are capable of down-regulating lymphocyte responses to mitogens and tumor-associated Ag by cell to cell contact interaction and increased PGE2 production. We have found that the tumor constitutively releases a granulocyte-macrophage (GM)-CSF-like factor in vivo and in vitro, which may be responsible for the systemic increase in cells of the macrophage lineage in tumor-bearing mice. A tumor cell line established from the in vivo tumor expresses and releases GM-CSF as shown by Northern and Western blot analyses. Daily i.p. injections for 3 wk of 10,000 U of rGM-CSF into normal mice induces hemopoietic and immunologic alterations similar to those observed in tumor bearers. Mac-1+ and/or Mac-2+ macrophages can also be detected in the spleens and bone marrow of the mice treated with rGM-CSF. Additionally, splenocytes from rGM-CSF-treated mice have reduced responses to mitogens and their peritoneal exudate cells can cause in vitro down-regulation of proliferative responses of lymphocytes from normal mice. The suppression can be partially reversed by the addition of indomethacin to the cultures suggesting that PGE2 may contribute to the effect. rGM-CSF enhances the in vitro release of PGE2 by the spleen, bone marrow, and peritoneal cells of normal mice. These data indicate that the high levels of GM-CSF constitutively produced by the tumor may be responsible for the hemopoietic changes and immunologic alterations observed in tumor-bearing mice.     

Neonatal tolerance induction in the thymus to MHC-class II-associated antigens. III. Significance of hemopoietic stem cells for induction and maintenance of Mls tolerance by continuous supply of tolerance-inducing nonlymphocytes

The role of hemopoietic stem cells and other cell types in the induction and maintenance of immunologic tolerance in the thymus was investigated by intravenous injection of Mls-semi-allogeneic cells into newborn mice less than 24 hr after birth. Mls-specific tolerance was induced by inoculation of peritoneal cells and thymus cells, and the tolerant state was compared with that induced by bone marrow cells which had hemopoietic stem cell activity and were able to create a stable chimera in both central and peripheral lymphoid organs. When peritoneal or thymus cells were injected, the level of tolerance attained was proportional to the number of cells injected, though peritoneal cells were 20 times as effective as thymus cells. In vivo functions of tolerance-inducing cells and their immediate precursors were radiosensitive and belonged to a Thy-1-, nylon-wool-nonadherent (probably non-B), weakly Sephadex G-10-adherent cell population. Tolerance induced by peritoneal cell injections was transient, starting to terminate within the first 2 weeks of life, while tolerance caused by bone marrow cell injections persisted through more than 6 weeks. Such transient tolerance induced by the former became long-lasting when followed by an additional injection of bone marrow cells, which did not cause thymic lymphocyte chimerism. All data indicated that bone marrow stem cells were engaged in tolerance induction and maintenance by continuously supplying tolerance-inducing nonlymphocytes.     

Intravenous endotoxin recruits a distinct subset of human neutrophils, defined by monoclonal antibody 31D8, from bone marrow to the peripheral circulation

Human neutrophils label with fluorochrome-labeled monoclonal antibody 31D8 as bright or dull. We determined the source and fate of 31D8 dull neutrophils by studying volunteers injected with endotoxin, epinephrine, or hydrocortisone, by examining bone marrow, and by examining skin blister exudate. We find that 31D8 dull neutrophils are normally not present in significant numbers in the circulation, are present in large numbers in normal marrow, and are recruited from the marrow by endotoxin, to a lesser extent by steroid, but not at all by epinephrine. 31D8 dull pattern correlates with morphologic immaturity in postendotoxin peripheral blood and bone marrow; however, blister exudate neutrophils contain only morphologically mature neutrophils, of which a significant number are 31D8 dull. We conclude that 31D8 dull neutrophils reside primarily in bone marrow and are released by agents which enhance bone marrow release of neutrophils. Their accumulation in skin blister exudate is unexplained, but suggests a special role in the inflammatory process.     

Activation of granulocyte cytotoxic function by purified mouse colony-stimulating factors

Highly purified mouse colony-stimulating factors (CSF) were tested for their effect on neutrophil cytotoxic function in a homologous antibody-dependent cell-mediated cytotoxicity (ADCC) assay in which TNP-coupled mouse thymoma cells coated with mouse anti-TNP antibodies were used as targets, and purified normal mouse bone marrow neutrophils or induced peritoneal neutrophils were used as effector cells. Biochemically pure granulocyte-macrophage (GM)- and granulocyte (G)-CSF enhanced the cytotoxic activity of neutrophils obtained from both sources, allowing them to kill target cells at low antibody concentrations. Furthermore, GM- and G-CSF showed an additive effect, suggesting either the presence of separate receptors for GM- and G-CSF or of separate subsets of neutrophils. Induced peritoneal neutrophils showed a higher level of basal cytotoxic activity than did bone marrow neutrophils, suggesting neutrophil activation in vivo, but both reached similar levels of cytotoxicity upon maximal stimulation with CSF. In addition, CSF was found to be cross-reactive between mouse and human species in their enhancement of neutrophil cytotoxicity. By testing purified mouse CSF on human neutrophils, it could be shown that G-CSF and GM-CSF are functionally distinct molecules, because only G-CSF enhanced ADCC by human neutrophils. These experiments show that the purified factors that control the production of neutrophils by progenitor cells in vitro also activate differentiated neutrophils to carry out their cytotoxic activity in a more effective manner.     

A lethal myeloproliferative syndrome in mice transplanted with bone marrow cells infected with a retrovirus expressing granulocyte-macrophage colony stimulating factor.

Murine bone marrow cells infected with a novel recombinant retrovirus, MPZen(GM-CSF), were engrafted into lethally irradiated recipients. The transplanted animals developed extremely high circulating levels of GM-CSF (up to 3 x 10(5) units/ml), and greatly elevated peripheral nucleated cell counts (up to 110 x 10(6) per ml). Their haemopoietic tissues contained GM-CSF proviral DNA and produced substantial levels of GM-CSF. The mice died within 4 weeks of transplantation with extensive neutrophil and macrophage infiltration of the spleen, lung, liver and peritoneal cavity and significant infiltration of both heart and skeletal muscle by neutrophils, macrophages and eosinophils. The thymus and lymph nodes were deficient in lymphoid cells. No disease occurred when infected cells from haemopoietic tissues of the primary transplanted animals were injected into normal or sub-lethally irradiated mice. Dysregulated GM-CSF expression by haemopoietic cells thus produces a fatal albeit non-neoplastic myeloproliferative syndrome.     

Colony formation in agar by adult bone marrow multipotential hemopoietic cells

Erythroid colony formation in agar cultures of CBA bone marrow cells was stimulated by the addition of pokeweed mitogen-stimulated spleen conditioned medium (SCM). Optimal colony numbers were obtained when cultures contained 20% fetal calf serum and concentrated spleen conditioned medium. By 7 days of incubation, large burst or unicentric erythroid colonies occurred at a maximum frequency of 40–50 per 10⁵ bone marrow cells. In CBA mice the cells forming erythroid colonies were also present in the spleen, peripheral blood, and within individual spleen colonies. A marked strain variation was noted with CBA mice having the highest levels of erythroid colony-forming cells. In CBA mice erythroid colony-forming cells were mainly non-cycling (12.5% reduction in colony numbers after incubation with hydroxyurea or 3H-thymidine). Erythroid colony-forming cells sedimented with a peak of 4.5 mm/hr, compared with CFU-S, which sedimented at 4.25 mm/hr. The addition of erythropoietin (up to 4 units) to cultures containing SCM did not alter the number or degree of hemoglobinisation of erythroid colonies. Analysis of the total number of erythroid colony-forming cells and CFU-S in 90 individual spleen colonies gave a correlation coefficient of r = 0.93 for these two cell types. In addition to benzidine-positive erythroid cells, up to 40% of the colonies contained, in addition, varying proportions of neutrophils, macrophages, eosinophils, and megakaryocytes. Taken together with the close correlation between the numbers of CFU-S in different adult hemopoietic tissues, including individual spleen colonies, the data indicate that the erythroid colony-forming cells expressing multiple hemopoietic differentiation are members of the hemopoietic multipotential stem cell compartment.     

The effect of cytokines on bactericidal activity of murine neutrophils

Abstract A range of recombinant cytokines have now been shown to modify aspects of the phenotype and function of human and murine neutrophils. However, few reports describe modification of the bactericidal activity of neutrophils. We therefore examined the recombinant murine cytokines tumor necrosis factor-α (TNF-α, 10–1000 ng ml⁻¹) and granulocyte macrophage-colony stimulating factor (GM-CSF, 10–1000 U ml⁻¹) for their ability to increase the bacterial killing capacity of murine neutrophils. Neutrophils from either bone marrow (fresh or cultured), or peritoneal exudates, or abscesses, were pre-incubated with either cytokine for 30–60 min and the killing of Proteus mirabilis, Escherichia coli , or Bacteriodes fragilis was examined in the presence or absence of serum over a 90 min period. Only for one combination was a small but significantly enhanced level of bacterial killing observed, the phagocytic killing of P. mirabilis by peritoneal exudate neutrophils in the presence of GM-CSF and serum. With this exception there was no enhancement of bacterial killing for the range of combinations of neutrophils and bacterial species tested. In contrast, at the concentrations tested for effect on bactericidal activity, TNF-α and GM-CSF were able to significantly upregulate CR3(but not FcγRII) expression on mouse neutrophils. There results indicate that upregulation of CR3 as an index of neutrophil activation does not necessarily correlate with increased bactericidal activity.     

Characterization of the priming effects of human granulocyte-macrophage colony-stimulating factor on human neutrophil leukotriene synthesis

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) is an in vitro and in vivo stimulator of human bone marrow myelomonocytic precursor cells and mature granulocyte and macrophage effector cells. We have compared the effect of GM-CSF on the synthesis of 5-lipoxygenase products induced by the chemotactic peptide fMet-Leu-Phe and the calcium ionophore A23187 in human neutrophils. Although GM-CSF alone did not stimulate detectable synthesis of products of the 5-lipoxygenase pathway, pre-incubation of neutrophils with 200 pM GM-CSF for 1 hour at 23 degrees C enhanced synthesis of leukotriene B4, its all-trans isomers and omega-oxidation products, and 5-hydroxyeicosatetraenoic acid in response to both the calcium ionophore A23187 (1.5 microM), and the chemotactic peptide fMet-Leu-Phe (0.1 microM). This priming effect of GM-CSF was maximal after a 60 min incubation at 23 degrees C, or after a 30 min preincubation at 37 degrees C. The effect of GM-CSF was maximal using a concentration of 1 nM. Enhancement of the leukotriene synthesis stimulated by A23187 was only observed when the cells were stimulated by the ionophore for periods of 3 minutes or less. In contrast, the enhancing effect of GM-CSF was still apparent when cells were exposed to fMet-Leu-Phe for as long as 15 minutes. Furthermore, the enhancing effect of GM-CSF was ablated when neutrophils were stimulated with A23187 and exogenous arachidonic acid. However, co-addition of exogenous arachidonic acid with fMet-Leu-Phe did not entirely mask the effect of GM-CSF. Possible mechanisms of action of GM-CSF are discussed.     

造血细胞活力冷冻损伤的可恢复性

人骨髓冻存后其造血祖细胞活力有一定程度下降,本研究对这种下降的可逆性作了初步观察。结果发现,用双层法和单层法作CFU-GM培养时,未冻存骨髓集落产率相近,冻存骨髓双层法的CFU-GM产率高于单层法。骨髓细胞用20％FM-CM、PHA-LYCM、PHA-PMCM预孵育2h后,分别测定其CFU-GM、BFU-E与CFU-Mix,发现这种孵育过程对未冻存骨髓的集落产率无明显影响,而冻存骨髓的集落产率在孵育后可升高(GEMmeg除外)。说明骨髓造血祖细胞对冻存的损伤反应不均一,部分受损细胞在一定条件下可以恢复其增殖活力。这对于用冻存骨髓作骨髓移植可能有一定意义。     

Studies on the effect of inflammation on the acute phase response using rat liver slices

Liver slices from control and inflamed rats were incubated in McCoy's medium and incorporation of [3H]leucine into liver and medium proteins and into albumin and alpha 1-acid glycoprotein was monitored over 48 hr. The release of the new acute phase reactant, sialyltransferase was also monitored in this system. Earlier observations in which liver slices were incubated for 6 hr showed that increased leucine incorporation into liver and medium proteins and alpha 1-acid glycoprotein, coupled with decreased incorporation into albumin, correlated with the acute phase response of these proteins. Increased incorporation of leucine into these proteins was found following 48 hr incubation in McCoy's medium showing that slices were able to express the changes characteristic of the acute phase response over this longer time period of incubation. Sialyltransferase was released into medium in a linear fashion up to 15 hr and continued to increase for 30 hr in this system; there was a substantial increase in release of enzyme activity from slices from inflamed rats when compared to controls. Monokine-conditioned medium prepared from peritoneal exudate cells isolated from rats at various times after lipopolysaccharide administration was used to induce the acute phase response by intraperitoneal injection. Slices were prepared from these rats and sialyltransferase release from slices was monitored. Monokines prepared from peritoneal exudate cells isolated from rats at about 30 hr were most effective in stimulating sialyltransferase release from liver slices.     

Effects of a murine mammary tumor on in vivo and in vitro hemopoiesis

The effects of an autologous transplanted mammary tumor (RIII-T3) on hemopoiesis in RIII mice are described. Tumor-bearing animals died 30 to 40 days after inoculation and displayed splenomegaly, extreme neutrophilia, and moderately increased monocyte levels in the spleen, peripheral blood, and bone marrow. The precursors of neutrophils and monocytes, granulocyte/macrophage colony-forming cells (GM-CFC) were elevated in the spleen, bone marrow, and peripheral blood. RIII-T3-conditioned medium stimulated bone marrow GM-CFC and caused the myelomonocytic cell line, WEHI-3B, to differentiate in vitro. The conditioned medium did not stimulate erythroid, megakaryocyte, or eosinophil colony formation. When conditioned medium was fractionated, two peaks of activity corresponding to GM-CSF and G-CSF were observed, suggesting that the extreme neutrophilia observed in tumor-bearing animals may result from chronic exposure of the hemopoietic system to these hemopoietic hormones.     

Studies on the mechanism of platelet-activating factor production in GM-CSF primed neutrophils: involvement of protein synthesis and phospholipase A2 activation

GM-CSF regulates the growth of hemopoietic progenitor cells, enhances the responsiveness of mature PMN and primes these cells for synthesis of leukotrienes and PAF in response to secondary stimuli. The biochemical requirements for PAF production in GM-CSF primed PMN was examined using different metabolic inhibitors. GM-CSF stimulates uridine incorporation into RNA and inhibitors for RNA and protein synthesis decrease PAF synthesis in our model. This suggests a role for gene expression and de novo synthesis of proteins in the action of GM-CSF. Different PLA2 inhibitors, including a 9 amino-acid peptide derived from a conserved region of the calpactin superfamily, decrease PAF production, indicating that in GM-CSF primed PMN the chemotactic peptide fMLP triggers lipid mediator synthesis by activating PLA2.     

Peritoneal exudate cells. I. Growth requirement of cells capable of forming colonies in soft agar

Mouse peritoneal exudate cells induced by thioglycollate medium can form colonies in soft agar with a plating efficiency of about 5% (0.6%–10%). Cells from an unstimulated peritoneal cavity form no colonies or have a plating efficiency of less than 0.001 %. These colony-forming cells from the peritoneal exudate are similar to bone marrow colony-forming cells in vitro in that they both require a substance(s) present in conditioned medium from L-cells or mouse embryo fibroblasts or the serum from endotoxin-treated mice for the initiation and the continuation of their growth. However, peritoneal exudate colony-forming cells have a much longer initial lag period (10–14 days) and can survive longer in the absence of L-cell conditioned medium than bone marrow colony-forming cells. Only mononuclear cells, presumably macrophages, are observed in peritoneal exudate colonies, whereas bone marrow cell colonies contain both polymorphonuclear cells and macrophages.     

Studies on human cord blood progenitor cells

Analysis of agar cultures throughout a 56-day period determined the concentration and cell cycle status of at least 4 different subclasses of hemopoietic colony forming cells (CFC) in human cord blood (CB). Although the concentration of CFC in CB was not significantly different from bone marrow (BM) in day-12 cultures, neutrophil colonies reached their peak on about day 23 in CB cultures and on day 12 in BM cultures. This suggests that the CFC in CB are more primitive than those in BM. In CB cultures, colonies of small cells contained predominantly neutrophils on day 14 and eosinophils on day 35, while the late developing (day 35) colonies of large cells contained mast-cell-like cells (MCL).     

Neutrophil maturation and activation determine anatomic site of clearance from circulation

The long-term disposition of circulating neutrophils and the site of disappearance from circulation remain unclear. We investigated neutrophil localization in mice using (111)In-labeled murine peripheral blood neutrophils, mature bone marrow neutrophils, and peritoneal exudate neutrophils to track in vivo localization of these different cell populations. Infused peripheral neutrophils were found to localize equally between liver and marrow sites by 4 h (31.2 +/- 1.9 vs. 31.9 +/- 1.8%), whereas exudate neutrophils predominantly localized to liver (42.0 +/- 1.1%) and marrow-derived neutrophils to the marrow (65.9 +/- 6.6%) where they were found to localize predominantly in the hematopoietic cords. Stimulation of marrow neutrophils before infusion caused a shift in localization from marrow to liver, and subsequent induction of an inflammatory site after infusion and marrow sequestration led to remobilization of infused marrow neutrophils but not of peripheral neutrophils. These results indicate that the marrow participates in removing neutrophils from circulation, with evidence supporting both storage and perhaps disposal functions. Furthermore, models for circulating neutrophil homeostasis should consider that the site of retention is governed by the maturation and activation states of the cell.