Colony stimulating factor-1 stimulates diacylglycerol generation in murine bone marrow-derived macrophages, but not in resident peritoneal macrophages

Colony stimulating factor-1 (CSF-1) stimulates DNA synthesis in murine bone marrow-derived macrophages (BMM); however, unlike BMM, murine resident peritoneal macrophages (RPM) undergo a poor proliferative response. It has previously been shown that phosphatidylinositol-4,5-bisphosphate hydrolysis is not associated with CSF-1 action in BMM. In this report we demonstrate that, despite a lack of inositol trisphosphate generation, CSF-1 transiently elevated both [3H]myristoyl- and [3H]arachidonyl-diacylglycerol (DAG) in BMM in a dose-dependent fashion. CSF-1 failed, however, to stimulate an increase in either species of DAG in RPM. Thus, DAG could be a second messenger for the proliferative action of CSF-1 in macrophages. Other mitogenic agents, 12-0-tetradecanoyl phorbol 13-acetate (TPA) and exogenous phospholipase C, also increased BMM levels of [3H]myristoyl- and [3H]arachidonyl-DAG. The nonmitogenic agents, lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha) and zymosan, had different effects on the generation of either species of DAG in BMM. LPS failed to elevate either form, TNF-alpha increased only [3H]arachidonyl-DAG, while zymosan stimulated levels of both species of DAG. It therefore appears that increased diacylglycerol generation may be necessary, but perhaps not sufficient, for macrophage proliferation.     

The interaction of 125I-colony-stimulating factor-1 with bone marrow-derived macrophages

The colony-stimulating factor, CSF-1, stimulates cultured quiescent murine bone marrow-derived macrophages (BMM) to enter DNA synthesis with a lag phase of 10-12 h. The binding, dissociation, internalization, and degradation of 125I-CSF-1 by BMM during the lag phase were investigated. Quiescent BMM express approximately 5 X 10(4) cell surface receptor sites/cell but contain additional cryptic sites (approximately 10(5)/cell) that can appear at the cell surface within 10 min at 37 degrees C. Studies of the binding reaction at both 2 degrees C (Kd less than or equal to 2 X 10(-13) M) and 37 degrees C (Kd approximately 4 X 10(-10) M) are consistent with the existence of a single class of cell surface sites. The disappearance of cell surface 125I-CSF-1 following a 2-37 degrees C temperature shift results from two, competitive, first order processes, internalization and dissociation. Internalization (t1/2 = 1.6 min) is 6 times more frequent than dissociation (t1/2 = 9.6 min). Following internalization, 10-15% of the intracellular CSF-1 is rapidly degraded whereas the remaining 85-90% is slowly degraded by a chloroquin-sensitive first order process (t1/2 greater than 3.5 h). These findings were confirmed and extended by studies of the uptake of 125I-CSF-1 at 37 degrees C. Following addition of 125I-CSF-1, cell surface receptors are rapidly down-regulated (t1/2 approximately 7 min) and their replacement does not commence until 20-60% of pre-existing surface receptor sites have disappeared. Despite receptor replacement, initially from the cryptic pool and later by de novo synthesis and/or receptor recycling (4 molecules/cell/s at steady state), the number of receptors at the cell surface remains low. The process results in the intracellular accumulation of large amounts of 125I-CSF-1 (greater than 10(5) molecules/cell) by BMM. Thus, whereas the kinetics of association, dissociation, and internalization of CSF-1 with BMM and peritoneal exudate macrophages are similar, BMM, which exhibit a higher proliferative response, degrade growth factor 12 times more slowly.     

CSF-1 stimulates glucose uptake in murine bone marrow-derived macrophages

3H-2-deoxyglucose was used as an isotopic tracer for the measurement of glucose uptake into quiescent murine bone marrow derived macrophages. A purified colony stimulating factor (CSF-1) was shown to stimulate 3H-2-deoxyglucose uptake in a dose-dependent manner. This stimulation was rapid, with a maximal effect seen at 20-30 minutes after growth factor addition. Both the inhibition by cytochalasin B and also the relative degree of competition by high concentrations of a series of glucose analogues suggest that the basal and CSF-1 stimulated 2-deoxyglucose uptake occur via a carrier facilitated D-glucose transport system. The data indicate that a purified growth factor can increase the glucose uptake in macrophages, a finding which could be relevant to the survival and/or the proliferative response of this and other haemopoietic cell types.     

Effects of granulocyte-macrophage colony-stimulating factor and colony-stimulating factor-1 on the proliferation and differentiation of murine alveolar macrophages

Murine alveolar macrophages (AM) were shown to have proliferative ability and to form colonies in vitro. The factors in lung-conditioned medium (CM) and L929-CM which stimulate the proliferation of AM were considered to be granulocyte-macrophage colony-stimulating factor (GM-CSF) and CSF-1, respectively, because recombinant murine (rm)GM-CSF and recombinant human (rh)CSF-1 could replace the activities of lung-CM and L929-CM, respectively. The phenotype of the cells in the colonies formed by AM incubated with rmGM-CSF or lung-CM was AM-like; more than 90% of the cells were stained by anti-asialo GM1 but not by FITC-LPS, and had AM-like morphology. Expression of Mac-1 Ag determined by M1/70HL in these cells as well as original AM was low. However, the phenotype of the cells in the colonies formed by AM incubated with rhCSF-1 or L929-CM was peritoneal macrophage (PM)-like; more than 90% of the cells were stained by FITC-LPS and M1/70HL, but not by anti-asialo GM1, and showed PM-like morphology. The cells in the colonies formed by AM incubated with rmGMCSF changed their phenotype after treatment with rhCSF-1; the percentage of cells stained by anti-asialo GM1 decreased, and that of cells stained by FITC-LPS increased. The cells in the colonies formed by AM incubated with rhCSF-1 never changed their phenotype after incubation with rmGM-CSF. In contrast to AM, more than 90% of the cells in all colonies formed by PM incubated with either rmGM-CSF, rhCSF-1, lung-CM, or L929-CM were stained by FITC-LPS but not by anti-asialo GM1. These results show that although AM and PM can proliferate, AM, in contrast to PM, are bipotential cells that can differentiate into two types of macrophages responding to distinct types of CSF, and that one of the molecular mechanisms controlling macrophage heterogeneity may be based on the type of CSF produced at distinct tissues.     

Different antigen presentation tendencies of granulocyte-macrophage colony-stimulating factor-induced bone marrow-derived macrophages and peritoneal macrophages

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced bone marrow-derived cells (BMCs) and primary peritoneal exudate cells (PECs) are usually used for antigen presentation in in vitro experiments. In order to expound their tendency for uptake and antigen presentation, we compared differences in the degree of phagocytosis, the expression of co-stimulatory molecules, and the activation of T lymphocytes between these two cell types. These assays used the F4/80 marker expression, as it is the general marker for macrophages. The BMC population was found to contain both F4/80(bright) and F4/80(dim) subtypes, while PECs were mainly composed of the F4/80(bright) subtype. Expression levels of cell surface co-stimulatory molecules, CD80, CD86, CD54, and CD40, were significantly higher for F4/80(+)BMCs than F4/80(+)PECs. Their expressions were further upregulated for F4/80(+)BMCs than for F4/80(+)PECs after stimulation with flagellin. F4/80(+)BMCs had a weaker ability to phagocytize microbeads than F4/80(+)PECs (P?相似文献   

Phorbol ester-stimulated superoxide production by murine bone marrow-derived macrophages requires preexposure to cytokines

Murine resident peritoneal macrophages (RPM) generate superoxide (O2-) in response to stimulation with PMA or zymosan. Murine bone marrow-derived macrophages (BMM) generate O2- in response to zymosan but not PMA. However, the ability to generate O2- in response to PMA could be induced in BMM by pre-exposing the cells to certain cytokines, including granulocyte-macrophage CSF (GM-CSF), tumor necrosis factor-alpha (TNF-alpha), IFN-gamma, and, to a lesser extent, IL-1 alpha. Bacterial LPS also induced the ability to respond to PMA. These same agents were also shown to prime RPM for enhanced PMA-induced respiratory burst. In contrast to GM-CSF, CSF-1 did not enhance the ability of BMM or RPM to generate O2- in response to PMA. Pretreatment with GM-CSF or TNF-alpha did not significantly affect the zymosan-induced release of O2- by BMM. These results suggest that unprimed BMM have a deficiency in the PMA-dependent signaling pathway that is corrected by exposure to selected cytokines. The results also raise the possibility that the basal ability of tissue macrophages to generate a respiratory burst in response to PMA may be a reflection of in vivo exposure to cytokines.     

Role of colony-stimulating factor-1 in bone metabolism

Colony-stimulating factor-1 (CSF-1) is a cytokine required for proliferation, differentiation, activity, and survival of cells of the mononuclear phagocytic system. The growth factor is synthesized as a soluble, matrix, or membrane associated molecule. The specific functions of these forms are not clear. However, some data suggest a dependence of the development of various populations of tissue macrophages on the locally expressed and presented cytokine. Deficiency in CSF-1, as is the case in the murine mutant strain op/op, results in low numbers of macrophages and monocytes and, most striking, leads to osteopetrosis due to a virtual absence of osteoclasts. Using the op/op mutation as a model, CSF-1 was established as one of the growth factors for osteoclasts. The expression of CSF-1 receptors, encoded by the proto-oncogene c-fms, by osteoclast precursors and osteoclasts, suggested an effect of this cytokine not only during osteoclast formation but also on the mature cells. In fact, CSF-1 was shown to inhibit the resorbing activity, to stimulate migration, and to support survival of isolated osteoclasts in vitro. By these actions on cells of the osteoclast lineage, CSF-1 induces recruitment of new osteoclasts, leading to a net increase of bone resorption, and might govern the spatial distribution of resorption sites within the bone. During these processes, locally expressed and presented forms of the growth factor may play a crucial role, as will be discussed in this article. © 1994 Wiley-Liss, Inc.     

GM-CSF and IL-3 stimulate diacylglycerol generation in murine bone marrow-derived macrophages

In murine bone marrow-derived macrophages, prelabeled with either [3H]myristic acid or [3H]arachidonic acid, the mitogenic colony stimulating factors GM-CSF and IL-3 stimulated a transient increase in [3H]diacylglycerol generation. Maximum [3H]diacylglycerol levels were detected at 10-15 min. The stimulation of [3H]diacylglycerol generation was dependent on the concentration of CSF and correlated with their ability to activate a variety of processes in the macrophage, including DNA synthesis. This is the first report to demonstrate that GM-CSF elevates diacylglycerol levels in macrophages and also to show that diacylglycerol generation may be an important signaling mechanism for IL-3 action. In conjunction with our recent demonstration that the mitogenic agents CSF-1, 12-0-tetradecanoylphorbol-13-acetate and exogenous phospholipase C also stimulate diacylglycerol generation in the macrophage (Veis and Hamilton, J.Cell.Physiol., 147, 298-305, 1991), our findings suggest that an increase in diacylglycerol levels is necessary but not sufficient for macrophage proliferation.     

Bacterial/CpG DNA down-modulates colony stimulating factor-1 receptor surface expression on murine bone marrow-derived macrophages with concomitant growth arrest and factor-independent survival

Unmethylated CpG motifs within bacterial DNA constitute a pathogen-associated molecular pattern recognized by the innate immune system. Many of the immunomodulatory functions of bacterial DNA can be ascribed to the ability to activate macrophages and dendritic cells. Here we show stimulatory DNA, like LPS, caused growth arrest of murine bone marrow-derived macrophages proliferating in CSF-1. Stimulatory DNA caused selective down-modulation of CSF-1 receptor surface expression. Flow cytometric analysis of CSF-1-deprived bone marrow-derived macrophages revealed that in contrast to the synchronous reduction of CSF-1 receptor upon CSF-1 addition, activating DNA (both bacterial DNA and CpG-containing oligonucleotide) caused rapid removal of receptor from individual cells leading to a bimodal distribution of surface expression at intermediate times or submaximal doses of stimulus. Despite causing growth arrest, both stimulatory DNA and LPS promoted factor-independent survival of bone marrow-derived macrophages, which was associated with phosphorylation of the mitogen-activated protein kinase family members, extracellular-regulated kinase 1 and 2. CSF-1 receptor down-modulation may polarize the professional APC compartment to the more immunostimulatory dendritic cell-like phenotype by suppressing terminal macrophage differentiation mediated by CSF-1.     

Colony-stimulating factor-1 modulates alpha 2-macroglobulin receptor expression in murine bone marrow macrophages.

Primary cultures of murine bone marrow macrophages (BMMs) were prepared from marrow cell suspensions. These cells expressed specific receptors that recognized the transformed conformation of human alpha 2-macroglobulin (alpha 2M) generated by reaction with CH3NH2. alpha 2M receptor expression was regulated by colony-stimulating factor-1 (CSF-1). The BMMs were deprived of CSF-1 for 6 h and then treated with different concentrations of the purified cytokine. After 18 h, binding of 125I-alpha 2M-CH3NH2 was examined at 4 degrees C. Analysis of the saturation isotherms and Scatchard transformations indicated that the KD was not affected by CSF-1 (1.9-2.4 nM), whereas the maximum specific radioligand binding capacity (Bmax) was increased from 5.6 x 10(4) receptors/cell in the absence of CSF-1 to 2.2 x 10(5) and 2.6 x 10(5) receptors/cell for BMMs treated with 1,000 and 10,000 units/ml CSF-1, respectively. The difference in total cellular protein after exposure to different levels of CSF-1 for 18 h was small (1.50-1.92 ng/cell) and not statistically significant. A 6-12-h lag phase was identified between the time of CSF-1 exposure and increased alpha 2M receptor expression. Cycloheximide completely blocked the increase in alpha 2M receptor expression when added simultaneously with the CSF-1; greater than 50% inhibition was observed when the cycloheximide was added up to 8 h later. The RNA synthesis inhibitors, actinomycin D and daunomycin, prevented increased alpha 2M receptor expression when added up to 4 h after the CSF-1, but had no effect at 8 h. At 37 degrees C, uptake and digestion of 125I-alpha 2M-CH3NH2 was increased in BMMs treated with 1,000 units/ml CSF-1 for 18 h compared with untreated cells. These studies demonstrate that CSF-1 increases the expression of alpha 2M receptors in BMMs through a pathway that requires new RNA and protein synthesis. We hypothesize that increased alpha 2M receptor expression may play an important role in cellular growth and differentiation.     

Biodegradable chitosan particles induce chemokine release and negligible arginase-1 activity compared to IL-4 in murine bone marrow-derived macrophages

Alternatively activated macrophages have been implicated in the therapeutic activity of biodegradable chitosan on wound healing, however, the mechanisms of phenotypic differentiation are still unclear.In vitro, macrophages stimulated with high doses of chitosan (?500 μg/mL) were reported to produce low-level markers associated with alternative activation (arginase-1) as well as classical activation (nitric oxide), and to undergo apoptosis. In this study, we tested the hypothesis that 40 kDa biodegradable chitosan (5–500 μg/mL) is sufficient to polarize mouse bone marrow-derived macrophages (BMDM) in vitro to an alternatively activated phenotype. Control cultures were stimulated with IL-4 (alternative activation), IFN-γ/LPS (classical activation), 1 μm diameter latex beads (phagocytosis), or left untreated. After 48 h of in vitro exposure, BMDM phagocytosed fluorescent chitosan particles or latex beads, and remained viable and metabolically active, although some cells detached with increasing chitosan and latex bead dosage. Arginase-1 was over 100-fold more strongly induced by IL-4 than by chitosan, which induced only sporadic and weak arginase-1 activity over untreated BMDM, and no nitric oxide. IFN-γ/LPS stimulated nitric oxide production and arginase-1 activity and high concentrations of inflammatory cytokines (IL-6, IL-1β, TNF-α, MIP-1α/MIP-1β), while latex beads stimulated nitric oxide and not arginase-1 activity. Chitosan or latex bead exposure, but not IL-4, tended to promote the release of several chemokines (MIP-1α/β, GM-CSF, RANTES, IL-1β), while all treatments promoted MCP-1 release. These data show that chitosan phagocytosis is not sufficient to polarize BMDM to the alternative or the classical pathway, suggesting that biodegradable chitosan elicits alternatively activated macrophages in vivo through indirect mechanisms.     

Transfection of immature murine bone marrow-derived dendritic cells with the granulocyte-macrophage colony-stimulating factor gene potently enhances their in vivo antigen-presenting capacity.

Ag presentation by dendritic cells (DC) is crucial for induction of primary T cell-mediated immune responses in vivo. Because DC culture from blood or bone marrow-derived progenitors is now clinically applicable, this study investigated the effectiveness of in vitro-generated murine bone marrow-derived DC (Bm-DC) for in vivo immunization protocols. Previous studies demonstrated that GM-CSF is an essential growth and differentiation factor for DC in culture and that in vivo administration of GM-CSF augments primary immune responses, which renders GM-CSF an attractive candidate to further enhance the effectiveness of DC-based immunotherapy protocols. Therefore, immature Bm-DC were transiently transfected with the GM-CSF gene and tested for differentiation, migration, and Ag-presenting capacity in vitro and in vivo. In vitro, GM-CSF gene-transfected Bm-DC were largely unaltered with regard to MHC and costimulatory molecule expression as well as alloantigen or peptide Ag-presenting capacity. When used for in vivo immunizations, however, the Ag-presenting capacity of GM-CSF gene-transfected Bm-DC was greatly enhanced compared with mock-transfected or untransfected cells, as determined by their effectiveness to induce primary immune reactions against hapten, protein Ag, and tumor Ag, respectively. Increased effectiveness in vivo correlated with the better migratory capacity of GM-CSF gene-transfected Bm-DC. These results show that GM-CSF gene transfection significantly enhances the capacity of DC to induce primary immune responses in vivo, which might also improve DC-based vaccines currently under clinical investigation.     

Macrophage colony-stimulating factor (M-CSF) enhances the capacity of murine macrophages to secrete oxygen reduction products

The capacity of macrophage colony-stimulating factor (M-CSF) to enhance respiratory burst activity in peritoneal macrophages was measured. Macrophages incubated for 48 hr or more with concentrated L cell-conditioned medium as a source of M-CSF released two to three times as much O2- in response to PMA as did unexposed macrophages. Stimulation was noted at concentrations of colony-stimulating activity from 0.1 to 2000 U/ml and was maximal at 10 to 100 U/ml. Purified, endotoxin-free CSF enhanced secretion to a similar degree as unpurified L cell-conditioned medium. Release of O2- by M-CSF macrophages occurred over 60 min and was triggered by opsonized zymosan as well as PMA. H2O2 release was also enhanced in macrophages exposed to both unpurified and purified M-CSF. These data indicate that M-CSF enhances the capacity of mature macrophages to release oxygen reduction products, and they are consistent with reports that CSF can stimulate the release of other secretory products.     

Modulation of colony-stimulating factor-1 receptors on macrophages by tumor necrosis factor

The effect of murine rTNF-alpha on the binding of human 125I-rCSF-1 to murine thioglycolate-elicited peritoneal exudate macrophages (PEM) was investigated. At 4 degrees C, 125I-CSF-1 binding to PEM was inhibited by preincubation with human rCSF-1, but not by other cytokines. When PEM were incubated with various cytokines at 37 degrees C, murine rTNF-alpha caused greater than 90% decrease in 125I-CSF-1 binding. This decrease was time, temperature and TNF dose dependent, and was not affected by preincubation with cycloheximide. The reduction in CSF-1-binding activity was reversed by prolonged incubation at 37 degrees C even in the presence of TNF. However, PEM preincubated with TNF subsequently washing free of residual TNF resulted in a rapid recovery of CSF-1 binding. This recovery of CSF-1-binding activity required protein synthesis. Binding studies suggested that the decrease in 125I-CSF-1 binding was most likely caused by a reduction in the number of CSF-1 receptors. In addition, preincubation with TNF at 37 degrees C inhibited 125I-CSF-1 binding on mononuclear phagocytes, including the macrophage cell line J774, bone marrow-derived macrophages, and nonelicited macrophages from three different strains of mice. In contrast, 125I-murine rTNF-alpha binding to PEM was not inhibited by preincubation with CSF-1 at 4 degrees C or 37 degrees C. These data suggest that TNF may play a role in the modulation of receptor expression on blood cells, and may point to a role for this pleiotropic cytokine in the regulation of hemopoiesis.     

Gamma interferon induces expression of Mad1 gene in macrophage, which inhibits colony-stimulating factor-1-dependent mitogenesis

Gamma interferon (IFN-gamma) has long been known as an antiproliferative cytokine. The mechanism of its action, however, remains elusive. Monocytes and macrophages are primary targets of IFN-gamma. To understand the antiproliferative signaling of IFNgamma, we studied the effect of IFNgamma on expression of c-Myc, Mad1, Max, cyclin D1, and cyclin D2 genes in both a macrophage cell line and in primary bone marrow-derived macrophages (BMM) in response to colony-stimulating factor-1 (CSF-1). We found that whereas IFNgamma inhibits CSF-1-stimulated c-Myc gene expression, it induces Mad1 expression. Induction of Mad1 mRNA could be detected as early as 90 min following IFNgamma treatment and was maintained for at least 15 h. These results suggest that IFNgamma treatment could shift the Myc-Max complex to the Mad1-Max complex in cells. The levels of Max, cyclin D1, and cyclin D2, however, remained unchanged. Enforced ectopic expression of Mad1 in the cells results in inhibition of [3H]thymidine incorporation and proliferation in response to CSF-1. This study suggests a mechanism by which IFNgamma inhibits CSF-1-stimulated proliferation of macrophages, i.e., by elevating the Mad1 level in the cells.     

Stromal cell-derived factor-1 promotes bone marrow-derived cells differentiation to cardiomyocyte phenotypes in vitro

Abstract.   Objective : Recent studies have demonstrated the potential of bone marrow-derived cells (BMDC) to differentiate into cardiomyocytes. Up-regulation of stromal cell-derived factor-1 (SDF-1), a member of the chemokine CXC subfamily, mediating recruitment of BMDC has been documented in infarcted myocardium; however, it remains unknown whether SDF-1 plays a role in cardiomyogenesis of BMDC. Materials and methods : Adherent BMDCs were cultured with SDF-1, or specific inhibitor for PI3K, CXCR4 or Akt with SDF-1, respectively. After 2 weeks, mRNAs and proteins from BMDCs were examined. Results : Two weeks after supplementation with SDF-1, either murine or human adherent BMDC cultured in vitro expressed cardiac specific mRNAs (NKX2.5, atrial natriuretic factor and heavy chain β-myosin) and proteins (troponin I and heavy chain cardiac myosin), and expression levels were partly decreased by combined treatment of CXCR4, PI3K or Akt inhibitor, with SDF-1. Conclusions : The novel findings suggest that beyond its role in mobilization and homing of BMDC, SDF-1 can promote BMDC to give rise to cardiomyocyte phenotypes in vitro , and the SDF-1/CXCR4/PI3K/Akt pathway may be one of the molecular mechanisms regulating cardiomyogenesis.     

Aggregated bovine IgG inhibits mannose receptor expression of murine bone marrow-derived macrophages via activation

We previously described the presence of an inhibitory protein contained in the 20 to 40% (NH4)2SO4 precipitable fraction of FCS that down-regulates expression of mannose receptors on bone marrow-derived macrophages. We now identify aggregated bovine IgG as the main inhibitory component. Heat-aggregated bovine IgG was capable of down-regulating expression of the macrophage mannose receptor in a dose-dependent manner without inducing changes in ligand affinity whereas neither F(ab')2 fragments nor nonaggregated IgG displayed any inhibitory effect. Depleting of IgG from heat inactivated FCS by protein G affinity chromatography completely removes the inhibitory activity. Moreover, readdition of the Ig eluate from the protein G chromatography column restored inhibition in a dose-dependent manner. Macrophages were able to clear exogenously added aggregated bovine IgG, thus leading to loss of inhibitory activity in macrophage-conditioned media as compared to sham-conditioned media containing aggregated IgG. These results indicate that aggregated IgG down-regulates mannose receptor expression by macrophage activation via interaction with Fc-gamma R.     

Colony-stimulating factor-1 induces thromboplastin activity in murine macrophages and human monocytes

Highly purified colony-stimulating factor-1 induced thromboplastin activity in murine macrophages and human monocytes in vitro. The activity increase was inhibited by cycloheximide and prevented by antibodies to CSF-1.