The development of macrophages from human CD34+ haematopoietic stem cells in serum-free cultures is optimized by IL-3 and SCF

The derivation of human macrophages from peripheral blood monocytes remains a convenient method for the study of macrophage biology. However, for macrophage differentiation, a significant proportion of development has occurred prior to the monocyte stage; monocyte subsets also have varying potential for differentiation. Differentiation of macrophages from a less mature precursor, such as CD34⁺ haematopoietic stem cells, can further inform with regard to the development of macrophage-lineage cells. CD34⁺ cells were cultured in serum-free medium containing Flt3L, SCF, IL-3, IL-6 and M-CSF. Using differing combinations of growth factors, the effect on cell proliferation and differentiation to adherent macrophage-like cells was determined. The proliferative response of CD34⁺ cells to M-CSF was determined during the initial phase of cell culture. Thirteen combinations of SCF, IL-3, IL-6 and M-CSF were then compared to determine the optimum combination for proliferation. Adherence was used to isolate mature macrophages, and the macrophage-like phenotype was confirmed by analyses of surface markers, histo-morphology and phagocytosis. This study refines the means by which large numbers of macrophages are obtained under serum-free conditions from haematopoietic precursors.     

Differentiation of the IL-3-dependent NFS-60 cell line and adaption to growth in macrophage colony-stimulating factor

Macrophage CSF (M-CSF) induces responsive bone marrow precursors into rapid growth and differentiation to mature macrophages. Available cell lines that depend on M-CSF for growth are well differentiated and rather adherent. We investigated the effects of M-CSF on immature myeloid cell lines as models of the marrow precursors. The murine line NFS-60 requires IL-3 for growth and also responds to granulocyte-CSF and granulocyte-macrophage-CSF. Cultures of one NFS-60 subline, when switched from IL-3 to 10% L cell conditioned media, a source of M-CSF, or purified M-CSF, frequently acquired large, adherent cells. The adherent cells grew slowly in the presence of M-CSF, in contrast to the majority population of small, round, rapidly growing cells. The large cells had properties of differentiated macrophages that were absent in the nonadherent cells. Cells with macrophage phenotype were not observed in IL-3-supported cultures over many passages. A subline was derived from NFS-60 that grew rapidly and continuously in human or murine M-CSF as round, nonadherent cells. The line, called M-NFS-60, responded well to M-CSF and IL-3, weakly to granulocyte-CSF and not at all to murine granulocyte-macrophage-CSF, IL-4, or human IL-1. A mAb to human M-CSF specifically inhibited only M-NFS-60 proliferation induced by the human growth factor, whether produced by mammalian or bacterial cells. This study shows two effects of M-CSF on the IL-3-dependent NFS-60 line. Upon first exposure to M-CSF, cells may undergo global differentiation to slowly replicating macrophages in conditions we have not been able to define. The more common effect is rapid growth of immature myeloid cells like the bone marrow precursors, but with a block to differentiation. Thus, these cells may be useful as models of M-CSF-induced differentiation, and of permanently maintained macrophage precursors.     

Regulation of osteoclast differentiation by the redox-dependent modulation of nuclear import of transcription factors

This study sought to characterize the reduced glutathione (GSH)/oxidized GSSG ratio during osteoclast differentiation and determine whether changes in the intracellular redox status regulate its differentiation through a RANKL-dependent signaling pathway. A progressive decrease of the GSH/GSSG ratio was observed during osteoclast differentiation, and the phenomenon was dependent on a decrease in total glutathione via downregulation of expression of the gamma-glutamylcysteinyl synthetase modifier gene. Glutathione depletion by L-buthionine-(S,R)-sulfoximine (BSO) was found to inhibit osteoclastogenesis by blocking nuclear import of NF-kappaB and AP-1 in RANKL-propagated signaling and bone pit formation by increasing BSO concentrations in mature osteoclasts. Furthermore, intraperitoneal injection of BSO in mice resulted in an increase in bone density and a decrease of the number of osteoclasts in bone. Conversely, glutathione repletion with either N-acetylcysteine or GSH enhanced osteoclastogenesis. These findings indicate that redox status decreases during osteoclast differentiation and that this modification directly regulates RANKL-induced osteoclastogenesis.     

Down-regulation of osteoprotegerin production in bone marrow macrophages by macrophage colony-stimulating factor

Macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-kappaB ligand (RANKL) induce the differentiation of bone marrow macrophages (BMMs) into osteoclasts. To delineate mechanisms involved, the effect of M-CSF on the production of osteoprotegerin (OPG), decoy receptor of RANKL, in BMMs was investigated. Mouse bone marrow cells were cultured with M-CSF for 4 days and adherent cells formed were used as BMMs. BMMs were cultured with or without M-CSF, and analyzed for expression of OPG and receptor activator of NF-kappaB (RANK; receptor for RANKL) mRNAs by real-time polymerase chain reaction and secretion of OPG by enzyme-linked immunosorbent assay. BMMs expressed macrophage markers, CD115 (c-fms), Mac-1 and F4/80, and showed phagocytotic activity. In addition, BMMs expressed OPG mRNA and secreted OPG into medium. M-CSF inhibited both the OPG mRNA expression and the OPG secretion dose-dependently and reversibly. The expression of RANK mRNA was not significantly affected by M-CSF. The results showed that M-CSF suppresses the OPG production in BMMs, which may increase the sensitivity of BMMs to RANKL.     

Plasticity of Human THP–1 Cell Phagocytic Activity during Macrophagic Differentiation

Studies of the role of macrophages in phagocytosis are of great theoretical and practical importance for understanding how these cells are involved in the organism’s defense response and in the development of various pathologies. Here we investigated phagocytic plasticity of THP–1 (acute monocytic human leukemia) cells at different stages (days 1, 3, and 7) of phorbol ester (PMA)–induced macrophage differentiation. Analysis of cytokine profiles showed that PMA at a concentration of 100 nM induced development of the proinflammatory macrophage population. The functional activity of macrophages was assessed on days 3 and 7 of differentiation using unlabeled latex beads and latex beads conjugated with ligands (gelatin, mannan, and IgG Fc fragment) that bind to the corresponding specific receptors. The general phagocytic activity increased significantly (1.5–2.0–fold) in the course of differentiation; phagocytosis occurred mostly through the Fc receptors, as shown previously for M1 macrophages. On day 7, the levels of phagocytosis of gelatin-and Fc–covered beads were high; however, the intensity of ingestion of mannan–conjugated beads via mannose receptors increased 2.5–3.0–fold as well, which indicated formation of cells with an alternative phenotype similar to that of M2 macrophages. Thus, the type and the plasticity of phagocytic activity at certain stages of macrophage differentiation can be associated with the formation of functionally mature morphological phenotype. This allows macrophages to exhibit their phagocytic potential in response to specific ligands. These data are of fundamental importance and can be used to develop therapeutic methods for correcting the M1/M2 macrophage ratio in an organism.     

Induction of macrophage colony-stimulating factor-dependent growth and differentiation after introduction of the murine c-fms gene into FDC-P1 cells.

A system has been established for analyzing the functions of the c-fms/macrophage colony-stimulating factor (M-CSF) receptor gene product in hematopoietic growth and differentiation. The murine c-fms gene was introduced into the factor-dependent murine hematopoietic cell line FDC-P1 by retroviral infection, and conversion to M-CSF-dependent growth was assayed in agar cultures. Expression of the c-fms gene in FDC-P1 cells, which normally do not express this gene, resulted in the conversion of resultant FD(c-fms) cells to M-CSF-dependent growth. Stimulation of FD(c-fms) cells by M-CSF led to the formation of colonies of altered morphology and produced reversible morphological changes suggestive of myeloid differentiation. M-CSF also induced expression of mature myeloid surface marker proteins in the FD(c-fms) cells. Neither multi-CSF nor granulocyte-macrophage CSF induced similar phenotypic changes but remained able to stimulate the proliferation of undifferentiated FD(c-fms) cells. These results indicate that the c-fms gene was expressed functionally in FDC-P1 cells and transmitted signals for growth. Also, the interaction of M-CSF with the c-fms gene product generated an additional signal for myeloid differentiation but did not irreversibly commit FD(c-fms) cells to terminal differentiation. This system can be used for molecular analysis of the growth- and differentiation-promoting activities of the c-fms proto-oncogene.     

Polycyclic aromatic hydrocarbons inhibit differentiation of human monocytes into macrophages

Polycyclic aromatic hydrocarbons (PAHs) such as benzo(a)pyrene (BP) are ubiquitous environmental carcinogenic contaminants exerting deleterious effects toward cells acting in the immune defense such as monocytic cells. To investigate the cellular basis involved, we have examined the consequences of PAH exposure on macrophagic differentiation of human blood monocytes. Treatment by BP markedly inhibited the formation of adherent macrophagic cells deriving from monocytes upon the action of either GM-CSF or M-CSF. Moreover, it reduced expression of macrophagic phenotypic markers such as CD71 and CD64 in GM-CSF-treated monocytic cells, without altering cell viability or inducing an apoptotic process. Exposure to BP also strongly altered functional properties characterizing macrophagic cells such as endocytosis, phagocytosis, LPS-triggered production of TNF-alpha and stimulation of allogeneic lymphocyte proliferation. Moreover, formation of adherent macrophagic cells was decreased in response to PAHs distinct from BP such as dimethylbenz(a)anthracene and 3-methylcholanthrene, which interact, like BP, with the arylhydrocarbon receptor (AhR) known to mediate many PAH effects. In contrast, benzo(e)pyrene, a PAH not activating AhR, had no effect. In addition, AhR was demonstrated to be present and functional in cultured monocytic cells, and the use of its antagonist alpha-naphtoflavone counteracted inhibitory effects of BP toward macrophagic differentiation. Overall, these data demonstrate that exposure to PAHs inhibits functional in vitro differentiation of blood monocytes into macrophages, likely through an AhR-dependent mechanism. Such an effect may contribute to the immunotoxicity of these environmental carcinogens owing to the crucial role played by macrophages in the immune defense.     

Altered differentiation in rat and rabbit limb bud micromass cultures by glutathione modulating agents

Glutathione (GSH) is the primary source of reducing equivalents in most cells, contributes significantly to the cellular redox potential and can control differentiation, proliferation, and apoptosis. Using limb bud micromass cultures from Sprague Dawley rats and New Zealand White rabbits, GSH modulating agents, L-buthionine-S,R-sulfoximine (BSO) and diethyl maleate (DEM) altered the formation of Alcian blue positive chondrogenic foci. Limb bud micromass cultures were treated for 5 d with BSO (50 or 100 μM) or DEM (5–25 μM). GSH content was determined by HPLC analysis. In rat cultures, BSO treatment did not affect differentiation but did show GSH depletion. In rabbit cultures, BSO completely inhibited differentiation and significantly depleted GSH. Treatment of rat cultures with DEM resulted in the dose-dependent decrease of chondrogenic foci, which correlated with a dose-dependent depletion of GSH. DEM completely inhibited rabbit limb bud cell differentiation and depleted GSH by 44%. Inhibition of differentiation was confirmed in rabbit cultures by the reduction in BMP-4 content. Addition of N-acetylcysteine to rabbit micromass cultures restored chondrogenic foci differentiation seen following treatment with both DEM and BSO. These results show species differences in GSH depletion in rat vs. rabbit limb bud cells and implicate GSH and cysteine in affecting pathways involved in chondrocyte differentiation.     

The network of hemopoietic regulatory proteins in myeloid cell differentiation.

There are clones of myeloid leukemic cells that can be induced to undergo terminal cell differentiation to macrophages by normal hemopoietic regulatory proteins. Induction of differentiation in two different clones of myeloid leukemic cells with interleukin 6 (IL-6) or granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in induction of mRNA for the hemopoietic regulatory proteins IL-6, GM-CSF, interleukin 1 alpha and interleukin 1 beta, tumor necrosis factor, and transforming growth factor beta 1. In one of these clones, induction of differentiation with GM-CSF was also associated with induction of mRNA for macrophage colony-stimulating factor (M-CSF) but not for the receptor for M-CSF (c-fms), whereas in the other clone, induction of differentiation with IL-6 was associated with induction of mRNA for both c-fms and M-CSF. The clones also differed in their responsiveness to these regulators. There was no induction of mRNA for granulocyte colony-stimulating factor or interleukin 3 during differentiation of either clone. The results indicate that the genes for a nearly normal network of positive and negative hemopoietic regulatory proteins are induced during differentiation of these myeloid leukemic cells and that there are leukemic clones with specific defects in this network.     

Fibroblast-secreted macrophage colony-stimulating factor is responsible for generation of biphenotypic B/macrophage cells from a subset of mouse B lymphocytes.

Normal and malignant CD5+ B lymphocytes can develop macrophage-like characteristics. One stimulus of this phenotypic shift is culture of normal mouse splenic B lymphocytes with splenic fibroblasts or their conditioned media. These biphenotypic B/macrophage (B/M phi) cells simultaneously display macrophage characteristics, such as phagocytosis and F4/80 expression, while retaining B cell features, including expression of surface Ig, CD5, B220, and rearranged Ig genes. The present study investigated the fibroblast-secreted factor that promotes this phenotypic change from B cell to B/M phi cell. RT-PCR analysis demonstrated that mRNA for M-CSF is produced by splenic fibroblasts. Recombinant M-CSF (CSF-1) could replace fibroblast-conditioned medium to elicit the development and survival of B/M phi cells from splenic B lymphocytes. In addition, neutralization of fibroblast-secreted M-CSF with specific mAbs abrogated the ability of conditioned supernatants to promote outgrowth of B/M phi cells. The transition from B lymphocyte to B/M phi cell was marked by the kinetic appearance of mRNA for the M-CSF receptor, c-fms, at day 3 following culture initiation. These results demonstrate that M-CSF is important in the development and physiology of mouse B/M phi cells and potentially in the growth of human biphenotypic hematological malignancies. Interestingly, the presence of IFN-gamma in splenic B lymphocyte cultures abrogated the effect of fibroblast-conditioned medium or M-CSF on outgrowth of B/M phi cells. Furthermore, these findings suggest that a Th1 microenvironment favored by typical macrophages is detrimental to the outgrowth of B/M phi cells.     

A possible role of glutathione and glutathione disulfide in tracheary element differentiation in the cultured mesophyll cells of Zinnia elegans.

We investigated the relationship between the cellular redox state of GSH or GSSG and tracheary element (TE) differentiation using a Zinnia experimental system, in which isolated mesophyll cells transdifferentiate to TEs. TE differentiation was suppressed by the application of L-buthionine sulfoximine (BSO), a potent inhibitor of GSH biosynthesis, at the early stage of cell culture. Application of GSSG at the early culture stage promoted the differentiation, but that of GSH or GSSG at an advanced period of culture suppressed the differentiation. Application of GSH and GSSG nullified the TE differentiation-suppressing effect of BSO. The results suggest that changes in the redox states of GSH and GSSG have a role in TE differentiation.     

P-selectin enhances generation of CD14+CD16+ dendritic-like cells and inhibits macrophage maturation from human peripheral blood monocytes

Endothelial cells play a critical role in monocyte differentiation. Platelets also affect terminal maturation of monocytes in vitro. P-selectin is an important adhesion molecule expressed on both endothelial cells and activated platelets. We investigated its effects on human peripheral blood monocyte differentiation under the influence of different cytokines. Generation of dendritic-like cells (DLCs) from peripheral blood monocytes was promoted by immobilized P-selectin in the presence of M-CSF and IL-4 as judged by dendritic cell (DC) morphology; increased expression of CD1a, a DC marker; low phagocytic activity; and high alloreactivity to naive T cells. In contrast to typical DCs, DLCs expressed CD14 and FcgammaRIII (CD16). These features link the possible identity of DLCs to that of an uncommon CD14(+)CD16(+)CD64(-) monocyte subset found to be expanded in a variety of pathological conditions. Functionally, DLCs generated by P-selectin in combination with M-CSF plus IL-4 primed naive allogeneic CD4(+) T cells to produce significantly less IFN-gamma than cells generated by BSA in the presence of M-CSF and IL-4. P-selectin effects on enhancing CD14(+)CD16(+) DLC generation were completely abrogated by pretreatment of cells with the protein kinase C delta inhibitor rottlerin, but not by classical protein kinase C inhibitor G?6976. Immobilized P-selectin also inhibited macrophage differentiation in response to M-CSF alone as demonstrated by morphology, phenotype, and phagocytosis analysis. The effects of P-selectin on macrophage differentiation were neutralized by pretreatment of monocytes with Ab against P-selectin glycoprotein ligand 1. These results suggest a novel role for P-selectin in regulating monocyte fate determination.     

IL-6 is a differentiation factor for M1 and WEHI-3B myeloid leukemic cells

IL-6 has multiple biologic activities in different cell systems including both the ability to support cell proliferation and to induce differentiation. We reported previously the isolation and functional expression of a mouse IL-6 (mIL-6) cDNA clone derived from bone marrow stromal cells. In this paper, we show that mIL-6 is a potent inducer of terminal macrophage differentiation for a mouse myeloid leukemic cell line, M1. Addition of mIL-6 to cultures of M1 cells rapidly inhibits their proliferation and induces phagocytic activity and morphologic changes characteristic of mature macrophages. These phenotypic changes are accompanied at the molecular level by a decrease in proto-oncogene c-myc mRNA accumulation and increases in Fc gamma R, proto-oncogenes c-fos and c-fms (CSF-1R) mRNA expression. Furthermore, IL-6 enhances the expression of Fc gamma R and c-fms in differentiation-responsive D+, but not unresponsive D- sublines of mouse myelomonocytic leukemic WEHI-3B cells. Together with our previous observation that IL-6 stimulates colony formation by normal myeloid progenitors, these results strongly suggest an important regulatory role for IL-6 in myeloid cell growth and differentiation.     

Quantitative analysis of phagocytosis of Cryptococcus neoformans by adherent phagocytic cells by fluorescence multi-well plate reader

Macrophages and monocytes are adherent phagocytic cells which play an important role in host defence against the yeast-like fungus Cryptococcus neoformans. Before, phagocytosis by adherent phagocytes could only be measured by means of microscopy or by a radioactive assay, which both have obvious disadvantages. We have developed a new, rapid and objective method to measure phagocytosis of C. neoformans by adherent phagocytes (e.g. alveolar macrophages) using a fluorescence multi-well plate reader. This method allows us to discriminate accurately between adherence and internalisation of C. neoformans by macrophages during long term incubation. In addition, the method was used to study the role of the mannose receptor in phagocytosis of the acapsular yeast in the absence of serum by human monocyte-derived macrophages (MDM).     

Role of glutathione depletion and reactive oxygen species generation in apoptotic signaling in a human B lymphoma cell line

The primary objective of this study was to determine the sequence of biochemical signaling events that occur after modulation of the cellular redox state in the B cell lymphoma line, PW, with emphasis on the role of mitochondrial signaling. L-Buthionine sulphoximine (BSO), which inhibits gamma glutamyl cysteine synthetase (gammaGCS), was used to modulate the cellular redox status. The sequence and role of mitochondrial events and downstream apoptotic signals and mediators was studied. After BSO treatment, there was an early decline in cellular glutathione (GSH), followed by an increase in reactive oxygen species (ROS) production, which induced a variety of apoptotic signals (detectable at different time points) in the absence of any external apoptotic stimuli. The sequence of biochemical events accompanying apoptosis included a 95% decrease in total GSH and a partial (25%) preservation of mitochondrial GSH, without a significant increase in ROS production at 24h. Early activation and nuclear translocation of the nuclear factor kappa B subunit Rel A was observed at approximately 3h after BSO treatment. Cytochrome c release into the cytosol was also seen after 24h of BSO treatment. p53 protein expression was unchanged after redox modulation for up to 72 h, and p21waf1 independent loss of cellular proliferation was observed. Surprisingly, a truncated form of p53 was expressed in a time-dependent manner, beginning at 24h after BSO incubation. Irreversible commitment to apoptosis occurred between 48 and 72 h after BSO treatment when mitochondrial GSH was depleted, and there was an increase in ROS production. Procaspase 3 protein levels showed a time-dependent reduction following incubation with BSO, notably after 48 h, that corresponded with increasing ROS levels. At 96 h, caspase 3 cleavage products were detectable. The pan-caspase inhibitor zVADfmk, partially blocked the induction of apoptosis at 48 h, and was ineffective after 72 h. PW cells could be rescued from apoptosis by removing them from BSO after up to 48, but not 72 h incubation with BSO. Mitochondrial transmembrane potential (DeltaPsi(m)) remained intact in most of the cells during the 72 h observation period, indicating that DeltaPsi(m) dissipation is not an early signal for the induction of redox dependent apoptosis in PW cells. These data suggest that a decrease in GSH alone can act as a potent early activator of apoptotic signaling. Increased ROS production following mitochondrial GSH depletion, represents a crucial event, which irreversibly commits PW cells to apoptosis.     

Differential effects of Trypanosoma brucei gambiense and Trypanosoma brucei brucei on rat macrophages

Mammalian immune responses to Trypanosoma brucei infection are important to control of the disease. In rats infected with T. brucei gambiense (Wellcome strain; WS) or T. brucei brucei (interleukin-tat 1.4 strain [ILS]), a marked increase in the number of macrophages in the spleen can be observed. However, the functional repercussions related to this expansion are not known. To help uncover the functional significance of macrophages in the context of trypanosome infection, we determined the mRNA levels of genes associated with an increase in macrophage number or macrophage function in WS- and ILS-infected rats and in cultured cells. Specifically, we assayed mRNA levels for macrophage colony stimulating factor (M-CSF), granulocyte macrophage colony stimulating factor (GM-CSF), and macrophage migration inhibitory factor (MIF). Upregulation of GM-CSF and MIF mRNA levels was robust in comparison with changes in M-CSF levels in ILS-infected rats. By contrast, upregulation of M-CSF was more robust in WS-infected rats. The phagocytic activity in macrophages harvested from ILS-infected rat spleens, but not WS-infected spleens, was higher than that in macrophages from uninfected rats. These results suggest that macrophages of WS-infected rats change to an immunosuppressive type. However, when WS or ILS is cocultured with spleen macrophages or HS-P cells, a cell line of rat macrophage origin, M-CSF is upregulated relative to GM-CSF and MIF in both cell types. Anemia occurs in ILS-, but not WS-infected, rats. Treatment of spleen macrophages or HS-P cells cocultured with ILS with cobalt chloride, which mimics the effects of anemia-induced hypoxia, led to downregulation of M-CSF mRNA levels, upregulation of GM-CSF and MIF, and an increase in phagocytic activity. However, the effect of cobalt chloride on spleen macrophages and HS-P cells cocultured with WS was restricted. These results suggest that anemia-induced hypoxia in ILS-infected rats stimulates the immune system and activates macrophages.     

Inhibition of Glutathione Production Induces Macrophage CD36 Expression and Enhances Cellular-oxidized Low Density Lipoprotein (oxLDL) Uptake

The glutathione (GSH)-dependent antioxidant system has been demonstrated to inhibit atherosclerosis. Macrophage CD36 uptakes oxidized low density lipoprotein (oxLDL) thereby facilitating foam cell formation and development of atherosclerosis. It remains unknown if GSH can influence macrophage CD36 expression and cellular oxLDL uptake directly. Herein we report that treatment of macrophages with l-buthionine-S,R-sulfoximine (BSO) decreased cellular GSH production and ratios of GSH to glutathione disulfide (GSH/GSSG) while increasing production of reactive oxygen species. Associated with decreased GSH levels, macrophage CD36 expression was increased, which resulted in enhanced cellular oxLDL uptake. In contrast, N-acetyl cysteine and antioxidant enzyme (catalase or superoxide dismutase) blocked BSO-induced CD36 expression as well as oxLDL uptake. In vivo, administration of mice with BSO increased CD36 expression in peritoneal macrophages and kidneys. BSO had no effect on CD36 mRNA expression and promoter activity but still induced CD36 protein expression in macrophages lacking peroxisome proliferator-activated receptor γ expression, suggesting it induced CD36 expression at the translational level. Indeed, we determined that BSO enhanced CD36 translational efficiency. Taken together, our study demonstrates that cellular GSH levels and GSH/GSSG status can regulate macrophage CD36 expression and cellular oxLDL uptake and demonstrate an important anti-atherogenic function of the GSH-dependent antioxidant system by providing a novel molecular mechanism.     

Colony-stimulating factor-1 receptor (c-fms)

The macrophage colony-stimulating factor, CSF-1 (M-CSF), is a homodimeric glycoprotein required for the lineage-specific growth of cells of the mononuclear phagocyte series. Apart from its role in stimulating the proliferation of bone marrow-derived precursors of monocytes and macrophages, CSF-1 acts as a survival factor and primes mature macrophages to carry out differentiated functions. Each of the actions of CSF-1 are mediated through its binding to a single class of high-affinity receptors expressed on monocytes, macrophages, and their committed progenitors. The CSF-1 receptor (CSF-1R) is encoded by the c-fms proto-oncogene, and is one of a family of growth factor receptors that exhibits an intrinsic tyrosine-specific protein kinase activity. Transduction of c-fms sequences as a viral oncogene (v-fms) in the McDonough (SM) and HZ-5 strains of feline sarcoma virus has resulted in alterations in receptor coding sequences that affect its activity as a tyrosine kinase and provide persistent signals for cell growth in the absence of its ligand. The genetic alterations in the c-fms gene that unmask its latent transforming potential abrogate its lineage-specific activity and enable v-fms to transform a variety of cells that do not normally express CSF-1 receptors.     

Expression of M-CSF receptor encoded by c-fms on smooth muscle cells derived from arteriosclerotic lesion.

Vascular smooth muscle cells in atherosclerotic lesions are phenotypically different from those in the normal arterial wall, and no expression of macrophage colony stimulating factor (M-CSF) receptor encoded by the proto-oncogene c-fms has been demonstrated in normal smooth muscle cells. In the present study, we demonstrated expression of c-fms and high affinity binding of M-CSF in smooth muscle cells isolated from an experimental rabbit model of arteriosclerosis (intimal smooth muscle cells), while no expression of c-fms was shown in medial smooth muscle cells. In the immunocytochemical analysis, both types of smooth muscle cells similarly reacted with an antibody specific to muscle cells (HHF 35) but did not react with an antibody specific to rabbit macrophages (RAM 11). In intimal smooth muscle cells, when cells were incubated with acetylated low density lipoproteins (LDL), the binding of acetylated LDL and foam cell formation were observed. In response to M-CSF, tyrosine-phosphorylation, as analyzed by the detection of anti-phosphotyrosine-reactive proteins, and an increased rate of cell proliferation were observed in intimal smooth muscle cells. These results indicated that intimal smooth muscle cells have the characteristics of monocyte-macrophages such as the expression of c-fms, which may be related to their proliferation and phenotypic conversion into foam cells in atheromatous lesions.