Murine Macrophage Colony-stimulating Factor Research Articles

Robust growth of avirulent phase II Coxiella burnetii in bone marrow-derived murine macrophages.

Published data show that murine bone marrow-derived macrophages (BMDM) restrict growth of avirulent phase II, but not virulent phase I, Coxiella burnetii. Growth restriction of phase II bacteria is thought to result from potentiated recognition of pathogen-associated molecular patterns, which leads to production of inhibitory effector molecules. Past studies have used conditioned medium from L-929 murine fibroblasts as a source of macrophage-colony stimulating factor (M-CSF) to promote differentiation of bone marrow-derived myeloid precursors into macrophages. However, uncharacterized components of conditioned medium, such as variable amounts of type I interferons, can affect macrophage activation status and their permissiveness for infection. In the current study, we show that the C. burnetii Nine Mile phase II (NMII) strain grows robustly in primary macrophages from C57BL/6J mice when bone marrow cells are differentiated with recombinant murine M-CSF (rmM-CSF). Bacteria were readily internalized by BMDM, and replicated within degradative, LAMP1-positive vacuoles to achieve roughly 3 logs of growth over 6 days. Uninfected BMDM did not appreciably express CD38 or Egr2, markers of classically (M1) and alternatively (M2) activated macrophages, respectively, nor did infection change the lack of polarization. In accordance with an M0 phenotype, infected BMDM produced moderate amounts of TNF and nitric oxide. Similar NMII growth results were obtained using C57BL/6J myeloid progenitors immortalized with an estrogen-regulated Hoxb8 (ER-Hoxb8) oncogene. To demonstrate the utility of the ER-Hoxb8 system, myeloid progenitors from natural resistance-associated macrophage protein 1 (Nramp1) C57BL/6J knock-in mice were transduced with ER-Hoxb8, and macrophages were derived from immortalized progenitors using rmM-CSF and infected with NMII. No difference in growth was observed when compared to macrophages from wild type mice, indicating depletion of metal ions by the Nramp1 transporter does not negatively impact NMII growth. Results with NMII were recapitulated in primary macrophages where C57BL/6J Nramp1+ BMDM efficiently killed Salmonella enterica serovar Typhimurium. M-CSF differentiated murine macrophages from bone marrow and conditional ER-Hoxb8 myeloid progenitors will be useful ex vivo models for studying Coxiella-macrophage interactions.

Proteomic Analysis Reveals Distinct Metabolic Differences Between Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) and Macrophage Colony Stimulating Factor (M-CSF) Grown Macrophages Derived from Murine Bone Marrow Cells*

Macrophages are crucial in controlling infectious agents and tissue homeostasis. Macrophages require a wide range of functional capabilities in order to fulfill distinct roles in our body, one being rapid and robust immune responses. To gain insight into macrophage plasticity and the key regulatory protein networks governing their specific functions, we performed quantitative analyses of the proteome and phosphoproteome of murine primary GM-CSF and M-CSF grown bone marrow derived macrophages (GM-BMMs and M-BMMs, respectively) using the latest isobaric tag based tandem mass tag (TMT) labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Strikingly, metabolic processes emerged as a major difference between these macrophages. Specifically, GM-BMMs show significant enrichment of proteins involving glycolysis, the mevalonate pathway, and nitrogen compound biosynthesis. This evidence of enhanced glycolytic capability in GM-BMMs is particularly significant regarding their pro-inflammatory responses, because increased production of cytokines upon LPS stimulation in GM-BMMs depends on their acute glycolytic capacity. In contrast, M-BMMs up-regulate proteins involved in endocytosis, which correlates with a tendency toward homeostatic functions such as scavenging cellular debris. Together, our data describes a proteomic network that underlies the pro-inflammatory actions of GM-BMMs as well as the homeostatic functions of M-BMMs.

Fluid-Phase Pinocytosis of Native Low Density Lipoprotein Promotes Murine M-CSF Differentiated Macrophage Foam Cell Formation

During atherosclerosis, low-density lipoprotein (LDL)-derived cholesterol accumulates in macrophages to form foam cells. Macrophage uptake of LDL promotes foam cell formation but the mechanism mediating this process is not clear. The present study investigates the mechanism of LDL uptake for macrophage colony-stimulating factor (M-CSF)-differentiated murine bone marrow-derived macrophages. LDL receptor-null (LDLR−/−) macrophages incubated with LDL showed non-saturable accumulation of cholesterol that did not down-regulate for the 24 h examined. Incubation of LDLR−/− macrophages with increasing concentrations of 125I-LDL showed non-saturable macrophage LDL uptake. A 20-fold excess of unlabeled LDL had no effect on 125I-LDL uptake by wild-type macrophages and genetic deletion of the macrophage scavenger receptors CD36 and SRA did not affect 125I-LDL uptake, showing that LDL uptake occurred by fluid-phase pinocytosis independently of receptors. Cholesterol accumulation was inhibited approximately 50% in wild-type and LDLR−/− mice treated with LY294002 or wortmannin, inhibitors of all classes of phosphoinositide 3-kinases (PI3K). Time-lapse, phase-contrast microscopy showed that macropinocytosis, an important fluid-phase uptake pathway in macrophages, was blocked almost completely by PI3K inhibition with wortmannin. Pharmacological inhibition of the class I PI3K isoforms alpha, beta, gamma or delta did not affect macrophage LDL-derived cholesterol accumulation or macropinocytosis. Furthermore, macrophages from mice expressing kinase-dead class I PI3K beta, gamma or delta isoforms showed no decrease in cholesterol accumulation or macropinocytosis when compared with wild-type macrophages. Thus, non-class I PI3K isoforms mediated macropinocytosis in these macrophages. Further characterization of the components necessary for LDL uptake, cholesterol accumulation, and macropinocytosis identified dynamin, microtubules, actin, and vacuolar type H(+)-ATPase as contributing to uptake. However, Pak1, Rac1, and Src-family kinases, which mediate fluid-phase pinocytosis in certain other cell types, were unnecessary. In conclusion, our findings provide evidence that targeting those components mediating macrophage macropinocytosis with inhibitors may be an effective strategy to limit macrophage accumulation of LDL-derived cholesterol in arteries.

Association of renal failure with poor response to PTH treatment in osteoporosis

Association of renal failure with poor response to PTH treatment in osteoporosis

Characterization of immortalized MARCO and SR-AI/II-deficient murine alveolar macrophage cell lines

BackgroundAlveolar macrophages (AM) avidly bind and ingest unopsonized inhaled particles and bacteria through class A scavenger receptors (SRAs) MARCO and SR-AI/II. Studies to characterize the function of these SRAs have used AMs from MARCO or SR-AI/II null mice, but this approach is limited by the relatively low yield of AMs. Moreover, studies using both MARCO and SR-AI/II-deficient (MS-/-) mice have not been reported yet. Hence, we sought to develop continuous cell lines from primary alveolar macrophages from MS-/- mice.ResultsWe used in vitro infection of the primary AMs with the J2 retrovirus carrying the v-raf and v-myc oncogenes. Following initial isolation in media supplemented with murine macrophage colony-stimulating factor (M-CSF), we subcloned three AM cell lines, designated ZK-1, ZK-2 and ZK-6. These cell lines grow well in RPMI-1640-10% FBS in the absence of M-CSF. These adherent but trypsin-sensitive cell lines have a doubling time of approximately 14 hours, exhibit typical macrophage morphology, and express macrophage-associated cell surface Mac-1 (CD11b) and F4/80 antigens. The cell lines show robust Fc-receptor dependent phagocytosis of opsonized red blood cells. Similar to freshly isolated AMs from MS-/- mice, the cell lines exhibit decreased phagocytosis of unopsonized titanium dioxide (TiO2), fluorescent latex beads and bacteria (Staphylococcus aureus) compared with the primary AMs from wild type (WT) C57BL/6 mice.ConclusionOur results indicated that three contiguous murine alveolar macrophage cell lines with MS-/- (ZK1, ZK2 and ZK6) were established successfully. These cell lines demonstrated macrophage morphology and functional activity. Interestingly, similar to freshly isolated AMs from MS-/- mice, the cell lines have a reduced, but not absent, ability to bind and ingest particles, with an altered pattern of blockade by scavenger receptor inhibitors. These cell lines will facilitate in vitro studies to further define MARCO and SR-AI/II function, and may also be useful to identify other novel scavenger-type macrophage receptors and for additional studies of particle toxicology.

Immortalization and Characterization of Murine Alveolar Macrophages with SRA‐I/II and MARCO‐knockout by a J2 Retrovirus

Alveolar macrophages (AM) avidly ingest unopsonized inhaled particles and bacteria through class A scavenger receptors (SRAs) SRA-I/II and MARCO. Studies to characterize the function of these SRAs have used AMs from SRA-I/II and MARCO knockout (KO) mice, but this approach is impeded by the relatively low yield of AMs. Hence, we sought to develop continuous cell lines from primary AMs from SRA-I/II and MARCO KO mice. We used in vitro infection with the J2 retrovirus carrying the v-raf and v-myc oncogenes. Following initial isolation in media supplemented with murine macrophage colony-stimulating factor, we subcloned three AM cell lines, designated ZK-1, ZK-2 and ZK-6. Their doubling time is approximately 12–16 hr grown in RPMI medium containing 10% FBS. All cell lines exhibit typical macrophage morphology upon microscopy after modified Wright’s stain and express macrophage-associated cell surface antigens. Similar to freshly isolated AMs from the KO mice, the cell lines exhibit decreased phagocytosis of unopsonized titanium dioxide, fluorescent latex beads and bacteria compared with the primary AMs from wild-type (Wt) mice. Unlike AMs from the Wt, only smaller 5-8-kDa dextran sulfate inhibited uptake of fluorescent beads. These cell lines will facilitate in vitro studies to further define SRA-I/II and MARCO function and may also be useful to identify other novel scavenger-type macrophage receptors.

Osteopetrotic Mouse Stroma with Thrombopoietin, c-kit Ligand, and flk-2 Ligand Supports Long-Term Mobilized CD34+Hematopoiesis In Vitro

OP-9 cells are stromal cells derived from macrophage colony-stimulating factor (M-CSF)-deficient osteopetrotic mice. To evaluate the OP-9 capability to sustain long-term hematopoiesis, we reported the expansion of granulocyte colony-stimulating factor (G-CSF)-mobilized human peripheral blood (PB) CD34(+) cells in co-culture with murine OP-9 and MS-5 stromal cells, either transfected with various combinations of adenovectors (Ad) expressing c-kit ligand (KL) (either soluble or transmembrane form), thrombopoietin (TPO), flt-3/flk2 ligand (FL), and granulocyte-macrophage (GM)-CSF or with weekly addition of these cytokines. Expression of TPO as well as association of TPO, FL, and KL increased progenitor cell and week-6 cobblestone area forming cell (CAFC) production in all stromal co-cultures. Similar progenitor expansion was obtained by weekly addition of soluble cytokine. Five weeks of co-culture with OP9 and TPO, FL + KL resulted in the greatest expansion of progenitor cells and week-6 CAFC as measured by secondary assay on MS-5. In contrast to MS-5 and TPO or TPO + FL + KL cultures where hematopoiesis declined by week 4, progenitor as well as week-6 CAFC expansion continued for over 3 months in TPO + FL + KL OP9 cocultures. This was associated with decrease of CD14(+) macrophage production. The addition of human macrophage (M)-CSF or CD14(+) cells to the co-culture decrease progenitor and stem cell expansion; however, murine M-CSF to OP-9 co-cultures did not decrease progenitor expansion. High levels of stromal-derived factor-1 (SDF-1) production by MS-5 and low or absent production by OP-9 may account for stem cell adhesion and CAFC formation in the former cultures and the predominance of stem and progenitor cells in the nonadherent fraction in the latter cultures.

Induction of potent antitumor response by vaccination with tumor lysate-pulsed macrophages engineered to secrete macrophage colony-stimulating factor and interferon-gamma.

Adoptive transfer of activated macrophages, being both effector cells and antigen-presenting cells, represents a promising approach to immunotherapy of cancer. In order to get activated macrophages with increased antitumor potential, in the present study, murine peritoneal macrophages were transduced with human macrophage colony-stimulating factor (M-CSF) and murine interferon-gamma (IFNgamma) by recombinant adenovirus infection. The results demonstrate that M-CSF and IFNgamma gene-modified macrophages exhibited higher expression of MHC-II, B7.1 and ICAM-1, increased antigen-presenting activity and cytotoxicity. It was also shown that they secreted more tumor necrosis factor, interleukin-1 and nitric oxide. In vivo experiments showed that in previously initiated murine pulmonary metastatic melanoma, tumor lysate-pulsed, M-CSF and IFNgamma gene-modified macrophages elicited more potent antitumor effects than tumor lysate pulsed M-CSF or IFNgamma gene-modified macrophages. Cytotoxic T lymphocyte (CTL) activity, IFNgamma and tumor-necrosis factor production of the splenocytes increased significantly in mice after intravenous injection of the gene-modified macrophages. M-CSF and IFNgamma gene-modified macrophages may act as activated effector and antigen-presenting cells, thus eliciting a more potent antitumor response.

Identification of Alternatively Spliced Transcripts Encoding Murine Macrophage Colony-Stimulating Factor

We have isolated a novel cDNA encoding macrophage colony-stimulating factor (M-CSF) from a murine stromal cell line, ST2. The cDNA included an entire coding sequence of the M-CSF gene but contained an additional sequence of 140 base pairs (bp). Northern blot analysis demonstrated that other murine cell lines such as a fibroblastic cell line (L) and a stromal cell line (PA6) also expressed the transcripts corresponding to the clone. The nucleotide sequence analyses of the cDNA and the cloned M-CSF genome revealed that the 140-bp insertion sequence was part of intron 1 which separated exon 1 and exon 2: the former contained part of the amino acid residues of the signal sequence and the latter the rest of the signal sequence and the first 22 amino acid residues of the mature protein. The insertion of the 140-bp intron sequence not only changed the amino acid sequence of the signal peptide but also generated an in-frame termination codon. However, instead of the dysfunction of the original initiation codon, the 140-bp insertion sequence contained a putative ATG initiation codon that preserved the original open reading frame. Finally, we found that the cDNA directed the expression of a secreted and biologically active M-CSF protein when it was introduced into COS7 cells and M-CSF activity in the culture supernatants was measured using an M-CSF-dependent cell line. These results indicate the presence of an alternatively spliced M-CSF transcript which utilizes an alternate initiation codon in order to specify active M-CSF protein.

Low-density lipoprotein stimulates the expression of macrophage colony-stimulating factor in glomerular mesangial cells

Disordered lipoprotein metabolism and the enhanced influx and accumulation of circulating mononuclear leukocytes into vascular tissue are common pathobiological phenomena associated with both atherosclerosis and glomerulosclerosis. Since atherogenic lipoproteins (such as low density lipoprotein, LDL) have been implicated in monocyte migration and proliferation into the glomerular mesangium, we examined the effect of LDL on mesangial cell expression of macrophage colony-stimulating factor (M-CSF), a cytoregulatory peptide associated with monocyte chemoattraction, differentiation and proliferation. Mesangial cell M-CSF gene expression, protein synthesis and secretion, and its biological activity to induce progenitor colony formation and monocyte proliferation were studied in murine mesangial cells. Incubation of either primary cultures or SV-40 transformed murine mesangial cells with LDL (0 to 200 micrograms/ml) induced M-CSF steady-state mRNA expression, in a dose-dependent manner (52 to 183% of control) when Northern blots were analyzed quantitatively by densitometric scanning. Similarly, Western blot analysis showed that LDL-activated SV-40 transformed mesangial cells increased M-CSF protein synthesis and secretion in a dose-dependent manner. The conditioned media obtained by incubating mesangial cells with LDL induced bone marrow progenitor colony formation that could be inhibited by specific neutralizing antibodies against murine M-CSF. Finally, the biological activity of M-CSF secreted by LDL-activated mesangial cells was further confirmed by its enhanced ability to induce monocyte proliferation. These data indicate that LDL, by activating mesangial cells to induce M-CSF and possibly other monocyte chemoattractants, may regulate the migration and proliferation of cells of mononuclear leukocytic origin into the mesangium supporting a pathobiological role for LDL in glomerular injury.

Cloning and Expression of Unique Murine Macrophage Colony-Stimulating Factor Transcripts

Cocultivation of cells from the gamma-irradiated D2XRII murine bone marrow stromal cell line with an interleukin-3/granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent hematopoietic progenitor cell line FDC-P1JL26 stimulates the emergence of factor-independent hematopoietic cell sublines. Several lines of evidence suggested that M-CSF or a protein antigenically related to M-CSF, termed leukemogenic stromal factor (LSF), that was expressed by D2XRII cells may have played a role in the emergence of the factor-independent sublines. In an effort to isolate a factor antigenically related to M-CSF, molecular clones were isolated from a D2XRII cDNA library that hybridized to a mouse M-CSF genetic probe. Two of these molecular clones, designated 60.8.2 and 6452, contained an 885-bp deletion in the M-CSF coding region. Such a cDNA clone has not been previously described in the mouse, but a cDNA clone homologous to it has been isolated from a human pancreatic tumor cell line, MIA-PaCa-2. Three transcripts (4.8, 3.4, and 1.8 kb) were detected that hybridized to an oligonucleotide probe that was specific to RNA transcripts containing the 60.8.2 deletion. The level of the 1.8-kb transcript was not detectably induced by ionizing irradiation; however, the levels of the 4.8-kb and 3.4-kb transcripts and two other M-CSF transcripts of sizes to 4.4 kb and 2.3 kb showed a 1.4- to 2.2-fold increase after gamma irradiation. Reverse transcription-polymerase chain reaction showed that the deletion-specific transcript(s) was detected in multiple mouse bone marrow stromal cell lines and in normal mouse tissues. The present studies establish the existence of an increased spectrum of murine M-CSF transcripts in bone marrow stromal cells and other tissues. This complexity of transcripts along with their increased accumulation after irradiation provides additional evidence for a role of proteins encoded by M-CSF transcripts in the response of bone marrow stromal cells to ionizing irradiation.

Macrophage colony-stimulating factor release and receptor expression in bone cells.

Colony-stimulating factors (CSF) may play a role in bone resorption. To examine whether osteoblasts secrete colony-stimulating activity (CSA) in response to parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHrP), conditioned medium (CM) from ROS 17/2.8 cells and primary rat osteoblasts were examined for induction of clonal growth of cultured rat bone marrow cells. Untreated cells constitutively secreted CSA, which increased with PTH and PTHrP treatment. The colonies formed were principally comprised of macrophages, and preincubation of CM with antiserum to murine macrophage colony-stimulating factor (M-CSF) neutralized most of the CSA, suggesting that the osteoblast-derived CSA was predominantly due to M-CSF. PTHrP treatment upregulated steady-state M-CSF mRNA levels. To investigate a paracrine role for M-CSF in bone we examined bone tissue and cells for the M-CSF receptor c-fms using immunohistochemical techniques and demonstrated staining of mature osteoclasts both in situ and after isolation. We conclude that M-CSF is responsible for the majority of the CSA released by PTH- and PTHrP-treated rat osteoblasts. In addition we identified CSF-1 receptor expression in mature osteoclasts. These data suggest that M-CSF is a mediator of osteoblast-osteoclast interaction in PTH- and PTHrP-induced bone resorption.

Culture of ovine bone marrow-derived macrophages and evidence for serum factors distinct from M-CSF contributing to their propagation in vitro.

An in vitro system allowing the culture of ovine bone marrow-derived macrophages (BMMs) is described. Bone marrow (BM) cells from the sternum of 4- to 9-month-old sheep were cultured in liquid suspension in hydrophobic bags with medium containing 20% autologous serum and 20% fetal calf serum (FCS). Cells with macrophage characteristics were positively selected and increased four- to five-fold between day (d) 0 and d18. Granulocytes and cells of lymphoid appearance including progenitor cells were negatively selected and were diminished 50-fold during this 18-d culture. The addition of macrophage colony-stimulating factor (M-CSF)-containing supernatants to liquid cultures did not significantly improve the yield of BMM in 18-d cultures. In contrast, cell survival at d6 and macrophage cell yield at d18 depended on the concentration and source of serum in the culture medium. FCS and 1:1 mixtures of FCS and autologous serum were superior to autologous serum alone. Analysis of growth requirements of ovine BMMs suggested that they are under more complex growth control than their murine counterparts. In an [3H]thymidine incorporation assay with BM cells collected at different times of culture, d3 or d4 BM cells responded to human recombinant M-CSF, human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF), bovine GM-CSF, murine M-CSF or murine M-CSF-containing supernatants, and bovine interleukin 1 beta (IL-1 beta) in decreasing order of magnitude. Likewise, pure murine BMM populations harvested at d6 responded to homologous GM-CSF, IL-3, and human or murine M-CSF. FCS did not stimulate the proliferation of murine BMMs (d6) and of ovine BM cells (d3 or d4). In contrast, ovine BM cells harvested at d12 responded to FCS by proliferation in a dose-dependent manner but failed to proliferate in the presence of human or murine M-CSF or M-CSF-containing supernatants of mouse and sheep fibroblasts containing mouse macrophage growth-promoting activity. Likewise, various cytokine-containing supernatants and recombinant cytokines (murine IL-3, murine and human GM-CSF, murine and bovine IL-1 beta) did not promote proliferation of ovine d12 BM cells to an extent greater than that achieved with 15% FCS alone. Thus, ovine BMM proliferation is under the control of at least two factors acting in sequence, M-CSF and an unidentified factor contained in FCS. The ovine BMM culture system may provide a model for the analysis of myelomonocytopoiesis in vitro.

Regulation of macrophage colony-stimulating factor in liver fat-storing cells by peptide growth factors.

Macrophage colony-stimulating factor (M-CSF) selectively promotes mononuclear phagocyte survival, proliferation, and differentiation. The production of this factor within the liver may be necessary to support the relatively long-term survival of circulating monocytes as they migrate into tissues and differentiate into macrophages. We studied the constitutive expression and the effects of platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), and epidermal growth factor (EGF) on M-CSF mRNA levels and secretion of M-CSF in murine liver fat-storing cells (FSC), vascular pericytes likely involved in the development of liver fibrosis. By Northern analysis, using a murine M-CSF cDNA, FSC constitutively express two major transcripts of 4.4 and 2.2 kb, similar to those detected in mouse L cells, used as a control. Exposure to 10 ng/ml PDGF or bFGF increased M-CSF mRNA levels. Peak effects were observed at 3 and 6 h for PDGF and bFGF, respectively, returning to baseline levels by 12 h. Under basal conditions, detectable amounts of M-CSF, measured by radioimmunoassay, were found in cell supernatants conditioned for 8 and 24 h. PDGF and bFGF markedly stimulated the release of M-CSF as early as 8 h, an effect persisting for at least 24 h. These findings suggest that liver FSC release M-CSF upon stimulation by PDGF and bFGF and may contribute to the activation of resident or infiltrating cells in inflammatory liver diseases.

Reassignment of the human macrophage colony stimulating factor gene to chromosome 1p13-21

Macrophage colony stimulating factor (CSF-1) is a member of a family of glycoproteins that are necessary for the normal proliferation and differentiation of myeloid progenitor cells. The human CSF-1 gene has previously been assigned to chromosome 5 using somatic cell hybrids, and further localized to 5q33 by in situ hybridization with a 3H labelled cDNA probe. However, the murine macrophage colony stimulating factor gene (csfm) has been localized to a region on mouse chromosome 3 which was previously shown to be syntenic with the proximal region of 1p and not 5q. Using a human genomic DNA clone that contains the CSF-1 gene, we have localized CSF-1 to chromosome 1p13-21 by fluorescence in situ hybridization. The reassignment of the CSF-1 gene argues against its involvement in myeloid disorders with deletions of the long arm of chromosome 5.

Peptide growth factors stimulate macrophage colony-stimulating factor in murine stromal cells

Bone marrow stromal cells influence hematopoiesis through cell-cell interaction and release of hematopoietic growth factors. Macrophage colony-stimulating factor (M-CSF) is constitutively produced by several murine and human stromal cell lines and is induced by inflammatory mediators such as interleukin-1 alpha or tumor necrosis factor-alpha (TNF-alpha) in a variety of mesenchymal cells. Other potentially important regulatory molecules such as platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), released by activated monocytes in response to inflammation, stimulate the growth of human stromal cells. However, the effect of these peptide mitogens on M-CSF expression in stromal cells has not been explored. In this study, we used TC-1 murine bone marrow-derived stromal cells that constitutively secrete M-CSF to determine the effect of PDGF and bFGF on cell proliferation and M-CSF gene expression. PDGF and bFGF, but not TNF- alpha, were potent mitogens for the TC-1 cells. Similar to mouse L cells, TC-1 murine stromal cells constitutively expressed two major mRNA transcripts of 4.4 and 2.2 kb that hybridized to a murine M-CSF cDNA. PDGF, bFGF, and TNF-alpha markedly stimulated the steady-state expression of M-CSF mRNA with different time-course kinetics. The increased expression of M-CSF mRNA was associated with enhanced secretion of M-CSF as determined by radioimmunoassay. These findings suggest that PDGF, bFGF, and TNF-alpha may regulate hematopoiesis indirectly through release of M-CSF by stromal cells and may modulate, at least in part, the hematopoietic response to inflammation.

Peptide Growth Factors Stimulate Macrophage Colony-Stimulating Factor in Murine Stromal Cells

Bone marrow stromal cells influence hematopoiesis through cell-cell interaction and release of hematopoietic growth factors. Macrophage colony-stimulating factor (M-CSF) is constitutively produced by several murine and human stromal cell lines and is induced by inflammatory mediators such as interleukin-1 alpha or tumor necrosis factor-alpha (TNF-alpha) in a variety of mesenchymal cells. Other potentially important regulatory molecules such as platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), released by activated monocytes in response to inflammation, stimulate the growth of human stromal cells. However, the effect of these peptide mitogens on M-CSF expression in stromal cells has not been explored. In this study, we used TC-1 murine bone marrow-derived stromal cells that constitutively secrete M-CSF to determine the effect of PDGF and bFGF on cell proliferation and M-CSF gene expression. PDGF and bFGF, but not TNF-alpha, were potent mitogens for the TC-1 cells. Similar to mouse L cells, TC-1 murine stromal cells constitutively expressed two major mRNA transcripts of 4.4 and 2.2 kb that hybridized to a murine M-CSF cDNA. PDGF, bFGF, and TNF-alpha markedly stimulated the steady-state expression of M-CSF mRNA with different time-course kinetics. The increased expression of M-CSF mRNA was associated with enhanced secretion of M-CSF as determined by radioimmunoassay. These findings suggest that PDGF, bFGF, and TNF-alpha may regulate hematopoiesis indirectly through release of M-CSF by stromal cells and may modulate, at least in part, the hematopoietic response to inflammation.

A sandwich enzyme-linked immunoadsorbent assay for detection of murine macrophage colony-stimulating factor (CSF-1)

A simple three-layer sandwich enzyme-linked immunoadsorbent assay (sandwich-ELISA) has been developed for murine macrophage colony-stimulating factor-1 (CSF-1) using the two monoclonal antibodies on which we recently reported (J. Immunol. (1988) 141, 483). The anti-CSF-1 monoclonal antibodies used in this assay recognize different epitopes of the same antigen, thereby permitting the detection of low amounts of CSF-1. This assay is specific to murine CSF-1. Recombinant human macrophage colony-stimulating factor, murine GM-CSF, or IL-3, either alone or together with CSF-1, does not interfere with the assay. The advantage of this assay over other reported immunoassays for CSF-1 is that radiolabeled or large quantities of purified CSF-1 are not required. This sandwich-ELISA compares favorbly with other assays in its rapidity, simplicity, and sensitivity.

Localization of the murine macrophage colony-stimulating factor gene to chromosome 3 using interspecific backcross analysis

The chromosomal location of the murine macrophage colony-stimulating factor ( Csfm) gene was determined by interspecific backcross analysis. We mapped Csfm to mouse chromosome 3, 2.5 cM distal to Ngfb and Nras and 1.3 cM proximal to Amy-2. CSFM maps to human chromosome 5q, while AMY2, NGFB, and NRAS map to human chromosome 1p. The chromosomal location of Csfm thus disrupts a previously identified conserved linkage group between mouse chromosome 3 and human chromosome 1. The location of Csfm also identifies yet another mouse chromosome that shares synteny with human chromosome 5q, a region involved in several different types of myeloid disease.

Demonstration of a Blood-Bone Marrow Barrier to Macrophage Colony-Stimulating Factor

Several previous studies suggested that murine macrophage colony-stimulating factor (CSF-1) might have impaired access to hematopoietic cells in the marrow. The apparent lack of hematopoietic responses to exogenous CSF and the finding of available or unoccupied CSF receptors despite saturating CSF levels in the serum led to studies of a potential blood-bone marrow barrier for this factor. Groups of mice were injected with pure unlabeled CSF-1 by either intravenous (IV) or intraperitoneal (IP) routes. Marrow and spleen cells were obtained at intervals after injection, held at 0 degree C, and assessed for changes in binding of 125I-CSF. Saturation of all available CSF receptors is achieved in vitro with 100 to 150 U CSF/mL. Despite achieving serum levels of 5,000 to 7,000 U/mL after IV injection of 25,000 units of CSF, less than 50% of the marrow receptors and less than 85% of the splenic receptors were saturated or downregulated. The decline in receptor availability was transient, with return of receptor sites in two to four hours. Increasing the IV dose to 125,000 units increased serum CSF values to approximately 20,000 U/mL and led to a virtual disappearance of available receptors for two to three hours. When administered IP, only approximately 40% of marrow and 80% of splenic receptors were affected for two hours. It was necessary to increase the dose of CSF to 250,000 units IP to saturate or downregulate receptors for three to four hours after injection. These observations indicate a marked blood-bone marrow barrier and lesser blood-spleen barrier for the transfer of serum CSF to responsive hematopoietic cells in vivo.