Granulocyte/macrophage colony-stimulating factor stimulates monocyte and tissue macrophage proliferation and enhances their responsiveness to macrophage colony-stimulating factor

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a specific humoral growth factor that stimulates both neutrophilic granulocyte and macrophage production by bone marrow hematopoietic progenitor cells. GM- CSF also stimulates the proliferation and clonal growth of both tissue macrophages and blood monocytes. Although at low concentrations GM-CSF was unable to support the long-term growth of tissue macrophages, it greatly enhanced their responsiveness to macrophage CSF (M-CSF, or CSF- 1). This effect was also observed by treating macrophages with GM-CSF for a short time. GM-CSF did not compete with M-CSF for binding to M- CSF receptors nor was it inactivated by treatment with anti-M-CSF antiserum. Treatment of tissue macrophages with GM-CSF led to a rapid but transient downregulation of M-CSF receptors; prolonged incubation at 37 degrees C, however, resulted in a restoration and upregulation of M-CSF receptors. Identical effects were observed with both native or recombinant GM-CSF. This study suggests that GM-CSF regulates tissue macrophage production by two modes of action: (a) direct stimulation of macrophage proliferation, and (b) enhancement of their responsiveness to M-CSF.     

巨噬细胞集落刺激因子对DEL细胞增殖作用的研究

目的 研究巨噬细胞集落刺激因子对动脉粥样硬化病变处伴随血管平滑肌细胞共同积聚的巨噬细胞的增殖作用及机制。方法 以能分化为巨噬细胞表型的ＤＥＬ细胞为离体细胞实验模型。将富含巨噬细胞集落刺激因子的１９２９细胞条件培养液或重组的人巨噬细胞集落刺激因子加入体外培养的处于静止期的ＤＥＬ细胞,以氚标胸腺嘧啶核苷或５－溴－悄嘧啶 核苷掺入法分别检测ＤＥＬ细胞ＤＮＡ的合成,另以Ｎｏｒｔｈｅｒｎ ｂｌｏｔ法检测ＤＥ     

Synergistic stimulation of macrophage proliferation by the monokines tumor necrosis factor-alpha and colony-stimulating factor 1

The effects of pure recombinant human tumor necrosis factor-alpha (TNF) on the CSF-1-stimulated proliferation of well-defined populations of murine macrophages are examined. Primary bone marrow-derived macrophages (BMM) from endotoxin-resistant C3H/HeJ mice were characterized for homogeneity in comparison with a cloned, growth factor-dependent macrophage cell line (S1) also derived from C3H/HeJ bone marrow cells. The mitogenic effects of each factor, alone and in combination, on the proliferation of both macrophage populations over a two-day culture period were studied. In contrast to CSF-1, TNF alone only slightly stimulated macrophage proliferation. However, the combination of CSF-1 and TNF stimulated proliferation of both primary BMM and S1 cells 1.5- to 2-fold greater than the sum of their predicted individual contributions. Such synergy was observed even at very high (plateau) levels of factors. TNF was found to transiently down-regulate CSF-1 receptor levels on both populations. Down-regulation was maximal at one hour; however, receptor numbers returned to initial, or greater, levels after 24 hours of incubation. Thus, TNF, an inducible monokine, greatly enhances the maximal mitogenic effects of CSF-1, an inducer of TNF production. These observations suggest an autocrine rule for TNF that involves synergy with (and perhaps obligatory cooperation with) CSF-1 in the regulation of macrophage proliferation.     

The human granulocyte/macrophage colony-stimulating factor receptor alpha2 isoform influences haemopoietic lineage commitment and divergence

A number of alternatively spliced isoforms of haemopoietic growth factor receptors (HGFRs) have been described, but their role in human haemopoiesis remains undetermined. We have investigated the relative expression of the alpha1 and alpha2 isoforms of human granulocyte/macrophage colony-stimulating factor receptor (hGM-CSFR) during haemopoietic cell differentiation, and have shown that both subunits are independently regulated during differentiation of CD34+ human haemopoietic progenitor cells. To further investigate these ex-vivo observations, we established a series of murine FDCP mix cell lines, which, as a consequence of the ectopic expression of alpha1 or alpha2 hGM-CSFR, demonstrated differential differentiation responses to hGM-CSF. In this model system, hGM-CSFR-alpha2-expressing cells showed increased hGM-CSF-mediated erythroid/megakaryocytic differentiation compared with hGM-CSFR-alpha1-expressing cells.     

Potential role of macrophage colony-stimulating factor in the proliferation of DEL cells.

The replication and activation of both vascular smooth muscle cells and macrophages, which have previously entered the arterial wall, are key events in the atherosclerotic process. The importance of macrophage colony-stimulating factor (MCSF) in control of the growth/proliferation of both cell types confers to this compound a central role in the development of vascular lesions. In order to gain insight into the mechanisms of macrophage proliferation, we investigated the effect of MCSF upon the proliferation of DEL cells. DEL cells constitute a monocyte/histiocytic cell line that differentiates along a macrophage lineage following exposure to phorbol ester. DEL cells constitutively express MCSF, and its receptor MCSFR is encoded by c-fms. We examined whether MCSF might play a role in the proliferation of cultured DEL cells. [3H]Thymidine or 5-bromo-2-deoxyuridine (BrdU) incorporation was measured following the addition of recombinant MCSF or L929 cell supernatant (as a source of MCSF) to quiescent DEL cells. In DEL cells, serum-free L929 cell supernatant induced DNA synthesis in a dose-dependent manner, and such an effect could be blunted by pretreatment of L929 cell supernatant with anti-mouse MCSF antibody. In these cells, DNA synthesis could also be triggered in a dose-dependent manner by the addition of recombinant human MCSF (rh MCSF) or thrombin. These findings clearly show that MCSF influences DEL cell proliferation and suggest an autocrine loop activation. They indicate that MCSF plays an important role in the development of vascular lesions, which occur during atherosclerotic progression.     

Recombinant tumor necrosis factor enhances the proliferative responsiveness of murine peripheral macrophages to macrophage colony-stimulating factor but inhibits their proliferative responsiveness to granulocyte-macrophage colony-stimulating factor

Tumor necrosis factor (TNF) is a protein produced by activated macrophages in response to endotoxin. The effect of recombinant murine TNF (rMuTNF) on the growth of murine tissue-derived macrophage colony-forming units (CFU-M) which are responsive to both macrophage and granulocyte-macrophage colony-stimulating factors (M-CSF and GM-CSF), was studied. TNF alone did not stimulate macrophage proliferation but did prolong their survival in vitro. The proliferative response of CFU-M to M-CSF, however, was greatly enhanced by the presence of TNF. The enhancement effect of TNF is dose-dependent, reaching a maximum at approximately 50 U/mL. In contrast, the proliferative responsiveness of CFU-M to GM-CSF was inhibited by the concurrent addition of rMuTNF. Both effects appear to be caused directly by rMuTNF, rather than by the secondary factor(s) produced by TNF-treated macrophages. TNF treatment also induced a transient downmodulation of M-CSF receptors in cultured macrophages and accelerated their uptake and use of exogenous M-CSF, which may account for, at least in part, the enhanced proliferative activity in response to M-CSF. Short-term treatment (24 hours) was not sufficient to induce either an enhancing or an inhibitory effect upon CFU-M. This study suggests an autoregulatory role for TNF in the production of mature tissue macrophages by selectively enhancing their proliferative response to lineage specific growth factor, M-CSF.     

Sources of murine macrophage colony-stimulating factor that also contain growth factors for primitive macrophage progenitor cells

A new growth factor (synergistic factor, SF) has recently been described, which, in combination with a macrophage CSF source, is able to stimulate the proliferation of primitive high-proliferative-potential macrophage-progenitor cells (HPP-CFC) in mouse bone marrow in culture. It has been found that the addition of supraoptimal amounts of the crude CSF sources, extracts of pregnant mouse uterus and embryo, media conditioned by L cells or the mouse mammary tumor cell line (EMT6), stimulated the proliferation of HPP-CFC in the absence of any added SF. These preparations thus appear to contain a factor with similar biological properties to those of SF. This conclusion is supported by the results obtained from Sephacryl S200 chromatography of these three CSF sources, which indicate that in all three cases fractions with apparent molecular weight (MW) greater than 68,000 contained the major portion of the CSF-1 activity, whereas fractions with MW less than 68,000 contained the SF-like activity, together with minor amounts of CSF-1 activity.     

FLT3 ligand can substitute for macrophage colony-stimulating factor in support of osteoclast differentiation and function.

Although bone resorption and osteoclast numbers are reduced in osteopetrotic (op/op) mice, osteoclasts are nevertheless present and functional, despite the absence of macrophage colony-stimulating factor (M-CSF). This suggests that alternative factors can partly compensate for the crucial actions of M-CSF in osteoclast induction. It was found that when nonadherent bone marrow cells were incubated in RANKL with Flt3 ligand (FL) without exogenous M-CSF, tartrate-resistance acid phosphatase (TRAP)-positive cells were formed, and bone resorption occurred. Without FL, only macrophagelike TRAP-negative cells were present. Granulocyte-macrophage CSF, stem cell factor, interleukin-3, and vascular endothelial growth factor could not similarly replace the need for M-CSF. TRAP-positive cell induction in FL was not due to synergy with M-CSF produced by the bone marrow cells themselves because FL also enabled their formation from the hemopoietic cells of op/op mice, which lack any M-CSF. FL appeared to substitute for M-CSF by supporting the differentiation of adherent cells that express mRNA for RANK and responsiveness to RANKL. To determine whether FL can account for the compensation for M-CSF deficiency that occurs in vivo, FL signaling was blockaded in op/op mice by the injection of soluble recombinant Flt3. It was found that the soluble receptor induced a substantial decrease in osteoclast number, strongly suggesting that FL is responsible for the partial compensation for M-CSF deficiency that occurs in these mice.     

Direct interaction of proteoglycan macrophage colony-stimulating factor and basic fibroblast growth factor

The proteoglycan form of macrophage colony-stimulating factor (PG-M- CSF), but not M-CSF with a molecular weight of 85 kD (85-kD M-CSF), bound to immobilized basic fibroblast growth factor (bFGF), and, conversely, bFGF bound to immobilized PG-M-CSF, but not to the 85-kD M- CSF. PG-M-CSF has an additional amino acid sequence at its carboxyl terminus (part of a precursor sequence that is removed in 85-kD M-CSF by proteolytic processing) and it has one or two chondroitin sulfate glycosaminoglycan chains at the carboxyl terminus. Enzymatic removal of the chondroitin sulfate chain from PG-M-CSF had no effect on the binding between PG-M-CSF and bFGF. Ligand blotting analysis with radioiodinated bFGF showed that bFGF specifically bound to the polypeptide that corresponded to the carboxyl terminus of PG-M-CSF and was produced in Escherichia coli transfected with its gene. The exogeneous addition of heparan sulfate, which has strong affinity for bFGF, efficiently inhibited the binding between PG-M-CSF and bFGF. These results show that PG-M-CSF binds bFGF through its carboxyl terminal peptide and that the binding sites for PG-M-CSF and heparan sulfate on bFGF are located close together. PG-M-CSF also significantly reduced the mitogenic action of bFGF on Balb/c 3T3 mouse fibroblastic cells. Therefore, we conclude that PG-M-CSF not only binds bFGF, but also neutralizes the activity of the growth factor.     

High blood levels of macrophage colony-stimulating factor in preeclampsia

In pregnancy, the decidual cells produce and secrete large amounts of macrophage colony-stimulating factor (M-CSF). M-CSF stimulates the proliferation and differentiation of trophoblasts. In addition, it stimulates them in a dose-dependent manner to produce certain hormones, such as human chorionic gonadotropin and human placental lactogen. Based on these facts, M-CSF is considered to be an essential cytokine for placental maintenance. Because placental dysfunction may sometimes result from preeclampsia, ascertaining blood M-CSF levels in preeclamptic patients would be of interest. The blood was collected from 33 subjects, of whom 19 were normal pregnant women and 14 were preeclamptic patients. The M-CSF level was determined by the sandwich enzyme-linked immunosorbent assay method using three antibodies. The investigators measured peripheral blood M-CSF levels in preeclamptic subjects and compared them with levels in subjects with normal pregnancies. This study showed that peripheral blood M-CSF levels were significantly higher in preeclamptic patients in the 30th and 38th weeks of pregnancy (P < .005). This is the first report concerning high M-CSF blood levels in preeclamptic patients.     

Possible role of tumor necrosis factor-alpha in erythropoietic suppression by endotoxin and granulocyte/macrophage colony-stimulating factor

Injection of bacterial endotoxin or granulocyte/macrophage colony-stimulating factor (GM-CSF) into exhypoxic polycythemic mice simultaneously with erythropoietin (EPO) suppressed erythroid cell formation, as monitored by ⁵⁹Fe incorporation into circulating red blood cells. This effect was dose-dependent and time-dependent. GM-CSF did not inhibit erythroid cell formation directly, as the antibody to the GM-CSF did not neutralize the effect of endotoxin, the inducer of GM-CSF. The suppression of both agents could be partially corrected by prior injection of a monoclonal antibody to tumor necrosis factor α (anti-TNFα). These results indicate that the suppression of EPO-induced erythroid cell formation by endotoxin and GM-CSF was due in part to the production of TNFα. © 1996 Wiley-Liss, Inc.