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Prepublished online as a Blood First Edition Paper on September 12, 2002; DOI 10.1182/blood-2002-02-0569.
PHAGOCYTES
From the Institute for Molecular Bioscience and ARC
Special Research Centre for Functional and Applied Genomics, University
of Queensland, Brisbane, Australia; the Albert Einstein
College of Medicine of Yeshiva University, Bronx, NY; The Canberra
Hospital, Woden, Australian Capital Territory, Australia;
and the Department of Molecular Genetics, The Ohio State University,
Columbus, OH.
The c-fms gene encodes the receptor for macrophage
colony-stimulating factor (CSF-1). The gene is expressed selectively in the macrophage and trophoblast cell lineages. Previous studies have
indicated that sequences in intron 2 control transcript elongation in
tissue-specific and regulated expression of c-fms. In
humans, an alternative promoter was implicated in expression of the
gene in trophoblasts. We show that in mice, c-fms
transcripts in trophoblasts initiate from multiple points within the
2-kilobase (kb) region flanking the first coding exon. A reporter gene
construct containing 3.5 kb of 5' flanking sequence and the downstream
intron 2 directed expression of enhanced green fluorescent protein
(EGFP) to both trophoblasts and macrophages. EGFP was detected in
trophoblasts from the earliest stage of implantation examined at
embryonic day 7.5. During embryonic development, EGFP highlighted the
large numbers of c-fms-positive macrophages, including
those that originate from the yolk sac. In adult mice, EGFP
location was consistent with known F4/80-positive macrophage
populations, including Langerhans cells of the skin, and permitted
convenient sorting of isolated tissue macrophages from disaggregated
tissue. Expression of EGFP in transgenic mice was dependent on intron 2 as no lines with detectable EGFP expression were obtained where either
all of intron 2 or a conserved enhancer element FIRE (the
Fms intronic regulatory element) was removed. We
have therefore defined the elements required to generate myeloid- and
trophoblast-specific transgenes as well as a model system for the study
of mononuclear phagocyte development and function.
(Blood. 2003;101:1155-1163) The differentiation of macrophages from bone marrow
progenitors requires the coordinated expression of many genes needed
for mature cell function. This process is controlled by the
lineage-specific growth factor, macrophage colony-stimulating factor
(CSF-1), which acts by binding to cell-surface receptors (CSF-1R)
encoded by the c-fms proto-oncogene.1
We2-4 and others5,6 have studied the
transcriptional regulation of the c-fms gene as a route to understanding lineage commitment in the macrophage lineage. The c-fms mRNA is detectable in the earliest yolk sac phagocytes
formed during mouse development, prior to many other markers, including the macrophage-restricted transcription factor PU.1, and the expression in the embryo and adult mouse is largely restricted to cells of the
macrophage lineage.7,8 The only other major site where the
c-fms gene is expressed is in placental
trophoblasts.9,10 In humans, there is a
trophoblast-specific promoter that lies at the 3' end of the
platelet-derived growth factor receptor- The murine exon 2 c-fms promoter was more active in
transient transfections of a macrophage cell line, RAW264, than in
untransformed fibroblasts,12 but it was also active in a
wide range of tumor cell lines that do not express the full-length
endogenous mRNA.3,13 Tumor cells in which the promoter was
active were shown to produce c-fms transcripts that
contained exon 2 and extended into the downstream intron 2, but did not
have detectable full-length c-fms mRNA. Inclusion of intron
2 in reporter gene constructs abolished reporter gene expression in
nonmacrophage tumor cells, but significant activity was retained in
RAW264 macrophages.3 We showed elsewhere that intron 2 contains a DNase 1 hypersensitive site we refer to as the
Fms intronic regulatory element (FIRE). FIRE was required for maximal expression of a reporter gene in stably-transfected RAW264
macrophages.4 The intronic sequence without FIRE actually profoundly suppressed reporter gene expression driven by the exon 2 promoter.4
In this paper, we show that the proximal promoter of c-fms
combined with the first intron directed consistent expression of the
enhanced green fluorescent protein (EGFP) reporter gene in the same
locations as the endogenous gene, and appropriate expression required
the intronic elements described previously. We demonstrate that the
fms-EGFP reporter gene provides a definitive marker for cells of the mononuclear phagocyte lineage throughout embryonic development, in the bone marrow and peripheral blood and in all adult
tissues. These findings provide a framework for the use of the
c-fms promoter in applications where targeted manipulation of macrophage or trophoblast differentiation in transgenic animals is desired.
Analysis of c-fms transcripts in placental RNA
To construct a representative placental library, poly
A+-selected e14 placental RNA was converted into cDNA using
both random and oligo dT priming. This cDNA was cloned into a For Northern blot analysis, 20 µg total RNA was separated by
formaldehyde-agarose gel electrophoresis, transferred to nylon filters,
and probed with a [32P]-dCTP-labeled cDNA probe specific
for exon 1 or 2 of c-fms. Samples included RNA from
trophoblastic stem cells (TS)14 (generously provided by Dr
Janet Rossant, Toronto, ON, Canada), a macrophage cell line
(BAC1.2F5),15 and e10 and e14 placentae, all of which are
c-fms-positive9,10 as well as the
c-fms-negative L-cell line and mouse embryo fibroblasts (MEF).
Plasmid constructs
Generation of transgenic mice Transgenic (TG) mice were generated at the Transgenic Animal Service of Queensland, Brisbane, Queensland, Australia (www.tasq.uq.edu.au) by injection of the transgenes into pronuclei of (C57BL/6 × CBA)F1 (BCBF1) fertilized eggs. TG mice generated were maintained under specific pathogen-free conditions. The integration of the transgenes was investigated by polymerase chain reaction (PCR) analysis of tail biopsy DNA, amplifying the EGFP gene by using primers 5'-CTGGTCGAGCTGGACGGCGACG-3' (forward) and 5'-CACGAACTCCAGCAGGACCATG-3' (reverse). The amplification temperatures were 1 minute at 95°C, 1 minute at 60°C, and 1 minute at 72°C for 25 cycles after an initial denaturing step of 5 minutes at 95°C. Southern blot analyses were also conducted on genomic DNAs of some mice for confirmation of genotype using standard protocols.Cells and tissue culture Thioglycollate-elicited peritoneal macrophages (TEPMs) were isolated from peritoneal cavities after intraperitoneal injections of 1 mL of 10% thioglycollate broth, followed by peritoneal lavage with phosphate-buffered saline (PBS) 3 to 4 days later. Pulmonary macrophages were obtained from broncho-alveolar lavage (BAL), whereas bone marrow-derived macrophages (BMMs) were obtained by isolation of bone marrow cells from the femurs of adult mice followed by differentiation of cells in complete RPMI medium (Invitrogen) supplemented with 10% fetal bovine serum (Serum Supreme, BioWhittaker, Walkersville, MD) and 2 mM L-glutamine (Glutamax, Invitrogen), 20 U/mL penicillin, 20 µg/mL streptomycin (Invitrogen), and 100 U/mL CSF-1 (Chiron) for 7 days.16 For dual-color fluorescence activated cell sorting (FACS) analysis of splenocytes (Figure 5A) using Mac-1 (CD11b) and F4/80 antibodies, spleen cells were mechanically disaggregated by mincing the tissues using sterile scalpel blades. Spleen adherent cells were enriched by overnight incubation on bacteriologic petri dishes, to which they adhere weakly, followed by removal of the tissue culture (TC) media and floating cells. The remaining adherent cells were harvested by squirting the petri dish surface with medium using a 20-mL syringe and an 18-gauge needle, and were subjected to FACS analysis after washing in PBS. For FACS analysis of tissue macrophages in spleen, liver, and lung, enzymatic digestions were performed with 0.1 U/mL Collagenase and 0.8 U/mL Dispase (Roche, Castle Hill, Australia) in PBS/20% fetal calf serum (FCS)7 for 1 hour at 37°C. Dispersed cells were washed in PBS and strained through 100-µm cell strainers (BD Falcon, Bedford, MA) and single cell suspensions were subjected to FACS. For isolation of cells from intestinal lamina propria, epithelial cells were washed in calcium- and magnesium-free Hanks balanced salt solution (Invitrogen) and minced tissues were enzymatically digested as described for other tissues above. Disaggregated cells were separated into low- and high-density fractions via centrifugation on Nycodenz (Nycomed Pharma, Norway) gradient prior to FACS analysis.17Trophoblast cell examination in tissue culture was conducted by
culturing the ectoplacental cone (EC)18 from e6.5
transgenic mice embryos. Briefly, the concepti were dissected free from
the decidua under a stereomicroscope, the EC was then cultured on each
well of 24-well TC plates, in modified Eagle medium (MEM) Flow cytometry Examination of EGFP+ cells by flow cytometry was done on a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) and the data were analyzed on CellQuest software (Becton Dickinson). Isolated macrophages were directly subjected to FACS analyses after being washed and resuspended on 1XPBS. For c-fms 2-color FACS analyses, isolated cells were washed once in ice-cold PBS containing 0.1% bovine serum albumin (BSA) and 0.1% NaN3. Nonspecific binding was blocked by incubation of cells in 0.1% goat serum for 15 minutes, followed by incubation with anti-c-fms antibody (1:100 dilution of hybridoma supernatant19) for 30 minutes. Cells were then washed with PBS containing 0.1% BSA and 0.1% NaN3, and incubated with goat anti-rat phycoerythrin (PE) F(ab')2 (Serotec, Oxford, United Kingdom) for 30 minutes. Cells were subsequently washed with PBS containing 0.1% BSA and 0.1% NaN3, resuspended, and analyzed by FACS. For Mac-1 and F4/80 staining, the same procedure was conducted, except that PE-conjugated anti-Mac-1 or anti-F4/80 (Serotec) antibodies were used. For peripheral blood mononuclear cell (PBMC) analysis, mature red cells were lysed with lysis buffer (155 mM NH4CL, 10 mM KHCO3, and 0.1 mM EDTA [ethylenediaminetetraacetic acid]) prior to antibody staining.Histology and EGFP examination Various tissues were directly fixed for 2 hours in 4% paraformaldehyde in PBS after dissections, followed by an overnight incubation in 18% sucrose at 4°C. On the following day, tissues were embedded in Tissue-Tek OCT compound (Sakura Finetechnical, Tokyo, Japan) and frozen in liquid nitrogen. Frozen sections (8 µm to 12 µm thick) were cut at 16°C with a LEICA cryostat model CM3050
(Leica Instrument, Germany), and mounted in DAKO fluorescent mounting
medium (DAKO, CA). The fluorescence of the EGFP was visualized under an
Olympus AX70 microscope (Olympus Australia, Melbourne) and pictures
were either taken using Kodak film or digitally acquired using Spot RT
Colour digital camera model 2.2.0 (Diagnostic Instruments, Sterling
Heights, MI). For Langerhans cell examination, mouse ears were
separated into dorsal and ventral halves with forceps. Dorsal ear
halves were incubated in 20 mM EDTA/PBS for 1 hour. Epidermal sheets
were then mounted on glass slides. For whole-mount embryo observations,
freshly dissected embryos were usually unfixed or fixed with 4%
paraformaldehyde and visualized in PBS under an inverted Olympus AX70
microscope. Yolk sac examination was performed in embryos derived from
nontransgenic mothers mated to transgenic males.
Start site analysis The region flanking the first coding exon (exon 2) in both mouse and human genes contains the transcription start site used in macrophages. Matrix alignment of the region flanking this exon in mouse (accession number AF290879) and human (U63963, X14 720) reveals no sequence homology outside of the 3' end of the PDGFR gene and the 500-bp flanking exon 2 (data not shown). This finding suggested that the exon 1 promoter identified in the human c-fms gene, immediately distal to the PDGFR locus,5,11 might not be conserved in the mouse.To determine whether mouse and human genes employ different
transcriptional mechanisms to express c-fms mRNA in
trophoblastic tissues, we performed systematic 5'RACE analyses on mouse
e14 placental RNA utilizing primers based on the known mouse exon 2 sequence. The products were sequenced and revealed a heterogeneous set
of spliced transcripts arising from the region from
Transgenic animal characterization The 7.2fms-EGFP was microinjected into mouse embryos by standard methods, and progeny were screened for incorporation of the transgene by PCR and Southern blot (data not shown) analyses. There were 6 transgenic lines produced, but one did not transmit the transgene. For all of the 7.2fms-EGFP transgenic lines, EGFP was detected readily in peritoneal, bone marrow-derived, and broncho-alveolar lavage macrophages (Figure 3B). The level of EGFP expression in macrophages from 3 lines tested by flow cytometry was remarkably consistent (Table 2).
Intron 2 as well as the FIRE region required for the activity of the transgene in vivo In stable transfections of the macrophage line RAW264, removal of the first intron greatly reduced EGFP expression, and removal of the FIRE sequence abolished it.4 To extend these observations, we produced 4 independent transgenic lines with constructs using the 3.5-kb promoter alone, and 6 independent lines with the 7.2-kb promoter without the FIRE sequence. In contrast to the thioglycollate-elicited peritoneal macrophages (TEPM) or BMMs from the 7.2fms-EGFP transgenic mice, those obtained from lines expressing EGFP from the 3.5-kb promoter exhibited fluorescence in a smaller number of cells and at a much lower level (Figure 3B, upper row and Table 2). On tissue sections of these transgenic mice, the level of EGFP was below the limits of detection (not shown). Deletion of the FIRE region completely abolished the activity of the transgene in these macrophage populations in each of the 6 transgenic lines examined (Figure 3B, lower row and Table 2). Again, there was no detectable expression of EGFP in tissue sections of any of the transgenic lines produced with the 7.2fms FIRE-EGFP transgene. The data indicate that the
intron, and particularly the FIRE sequence, is absolutely needed for
the reproducible, position-independent expression, obtained with the
7.2fms-EGFP transgene.
c-fms/EGFP transgene is active in trophoblasts The analysis of the start site above suggests that the 3.5-kb fms promoter could direct trophoblast expression. In order to assess the possible activities of the c-fms promoter driving the EGFP reporter gene in these cells, EGFP expression in early embryonic development was determined. Fluorescence microscope observation of 7.2fms-EGFP TG embryos revealed the presence of green-fluorescent cells in the ectoplacental cone area of e7.5 embryos. Later in development, trophoblasts in the deciduum surrounding the embryo were also positive for EGFP, as were trophoblastic giant cells in e10.5 and e12.5 embryos (not shown). The locations of the EGFP+ cells in these embryos were similar to the expression of c-fms mRNA detected by in situ hybridization.8,20 The expression of EGFP in trophoblasts was also observed in giant cells grown in tissue-culture plates derived from the ectoplacental cones (Figure 4A). These observations support the data from the start site analysis, and demonstrated that the exon 2 c-fms upstream promoter region in the mouse is used by both macrophage lineage and trophoblast cells.
EGFP is widely expressed in phagocytic cells in tissues of the transgenic embryos Our laboratory has previously described the analysis of c-fms gene expression by whole-mount in situ hybridization in mouse embryonic development.7,8 The ability of the 7.2fms-EGFP transgene to recapitulate c-fms mRNA expression was examined systematically. The first c-fms-expressing cells were detected in the yolk sac around e9.5 and the location is recapitulated by EGFP+ cells. Their abundance and distribution is striking. They are not focused in blood islands, but distributed throughout the yolk sac (Figure 4B). There is no possibility that the green-fluorescent cells observed in the yolk sac originated from infiltration of cells of maternal origin because all the embryos examined were derived from nontransgenic mothers mated with a transgenic male. The origin and function of these yolk sac-derived embryonic phagocytes has been reviewed recently.21 By e10 to e10.5, significant numbers of EGFP+ cells were observed infiltrating first into the head, then the liver of the embryo (not shown), again entirely consistent with the published pattern of c-fms mRNA expression7 and independent evidence of the infiltration of hematopoietic progenitors and stem cells into the liver which occurs at e10.5 to e11.22 The number of green-fluorescent cells escalated rapidly through development of the embryo to e12 to e13. In particular, c-fms-positive cells have been shown to be involved in removing apoptotic cells in the interdigital regions in developing footplates of the mouse embryo.7,8 Similarly, Figure 4C shows massive accumulation of green-fluorescent cells in the interdigital regions at e12.5.Embryo sections at e13.5 revealed the presence of green-fluorescent cells throughout the body of the embryos, with extensive accumulation of the cells in regions with high tissue turnover and extensive cell death. Figure 4 shows some representative sites of high numbers of EGFP+ cell infiltration, including the epidermal layer of the dorsal part of the embryo (Figure 4D), as well as in the mesenchymal area beneath the chest (Figure 4E) and other surfaces of the body, the liver and its surrounding area (Figure 4F), and the brain. In the brain, EGFP+ cells were especially prevalent in the pons-midbrain junction area (Figure 4G), as well as in the choroid plexus (Figure 4H). Elsewhere in the embryo, numerous green-fluorescent cells were found in the mesenchymal area around the somites (Figure 4I), lung (Figure 4J), around the intrinsic muscles of the tongue (Figure 4K), and also in the developing eye (Figure 4L). Most importantly, the morphology, abundance, and location of EGFP+ cells are consistent with absolute restriction of the transgene to embryonic phagocytes, and to the known location of cells expressing c-fms mRNA and other macrophage-specific genes.7,8 EGFP is specifically expressed in myeloid cells in the bone marrow, blood, and tissues Mononuclear phagocytes develop from bone marrow progenitors, enter the circulation as blood monocytes, and then leave to replenish tissue macrophage populations. Macrophages share progenitors with granulocytes, and the knockout of the PU.1 transcriptional regulator,23 which affects transcription of c-fms,24 has deficiencies in both lineages. Although c-fms mRNA and protein are absent from mature granulocytes, the more stable EGFP reporter could be retained in progeny from the common precursor. Dual-color fluorescence immunostaining was carried out on bone marrow and peripheral blood using the F4/80 and Mac-1 (CD11b) markers. Additionally, we examined CSF-1R (c-Fms) surface protein expression. Representative profiles are shown in Figure 5B. In bone marrow, most EGFP-expressing cells also expressed CD11b, indicating that the transgene expression is restricted to myeloid cells. By contrast, few CD11b cells were EGFP+. About 50% of the
EGFP+ cells also expressed the mature macrophage marker
F4/80 and/or detectable surface CSF-1R. Among peripheral blood
mononuclear cells, the EGFP+ cells all expressed both F4/80
and CD11b, and the proportion upon which surface CSF-1R was
undetectable was smaller than in marrow (Figure 5B). Figure 5C shows
the expression of CD11b and F4/80 on EGFP+ peritoneal and
broncho-alveolar lavage cells. All EGFP+ cells in the
peritoneum express both markers. By contrast, alveolar macrophages, as
expected,25 lack CD11b but express F4/80 at low levels.
The analysis of alveolar macophages is complicated by the relatively
high background autofluorescence.
EGFP is expressed in tissue macrophage populations Macrophages defined by the F4/80 surface marker in the mouse are a major component (10%-15%) of the cells in most tissues of the body.26-32 The images of F4/80 location have been loaded in a database at www.imb.uq.edu.au/groups/hume. F4/80 is not detected on all putative tissue macrophages, and, as evident from Figure 5C, is especially low in lung macrophages and macrophages of lymphoid organs. EGFP was detected in all of the major macrophage populations of the body. In the spleen, the red pulp macrophages were strongly EGFP+ (Figure 6A). Enzymatic digestion of the spleen followed by flow cytometry revealed approximately 18% of cells that were EGFP+. The majority of these cells adhered to bacteriologic plastic, supporting their identity as macrophages (Figure 5A). Similarly, in the liver EGFP was expressed specifically on sinusoidal stellate cells, resembling liver macrophages (Kupffer cells) with the characteristic concentration toward the periphery of liver lobules. In this organ, these cells constituted about 8% to 9% of the whole liver cells, as examined by FACS analysis following enzymatic disaggregation (Figure 6B). In the lung there is a large interstitial macrophage population in addition to those present in broncho-alveolar spaces.33 EGFP expression was detected on 8% of cells from enzymatically digested lung (Figure 6C). Finally, in the intestinal lamina propria there is a large population of macrophages that can be isolated following enzymatic digestion. Figure 6D shows the tissue section and the FACS profile of the enzymatically digested mouse intestinal wall, where 6% to 8% of cells were EGFP+. Nycodenz gradient centrifugation of this digested tissue could increase the proportion of EGFP+ cells up to 15%, consistent with previous studies on purification of these cells.17
Macrophages and c-fms expression in the brain are of particular interest because of the neurologic abnormalities in the CSF-1-deficient Csf1op/Csf1op mouse34 and the isolated claim that the human c-fms promoter drives transgene (lacZ) expression in astrocytes.35 The F4/80 antigen is readily detected on brain macrophages (microglial cells).36 In the brain, EGFP was detectable in macrophages associated with the microvasculature and meningeal surfaces (not shown) and in microglia (Figure 6E). In this organ, microglial processes were clearly evident and highlighted by the fluorescent protein expression. Expression of EGFP in microglia can also be clearly observed in the retina, where they spread in 2 dimensions in the plane of the inner and outer plexiform layers28 (Figure 6F). In neither brain nor retina was there any evidence of expression in glial cells other than microglia. Macrophages defined by F4/80 antigen expression are abundantly associated with epithelia and in endocrine organs where they may perform specific physiologic functions.26,30,37 In an extensive survey, EGFP expression was generally consistent with previous data on F4/80. Examples shown include the lamina propria of the gastrointestinal tract (Figure 6D), the renal medulla (Figure 6G), and interstitium of the testis (Figure 6H). A number of additional examples and further images are displayed at www.imb.uq.edu.au/groups/hume. Bone resorptive osteoclasts share a progenitor with macrophages and their production is CSF-1 dependent. Although they lose mature macrophage markers such as F4/80,29 mature osteoclasts express c-fms mRNA and CSF-1 can acutely regulate osteoclastic bone resorption.38-40 In the transgenic mice, osteoclasts expressed detectable EGFP both in culture and bone sections (Figure 6I). EGFP expression in lymphoid organs and dendritic cells Cell populations such as the Langerhans cells (LCs) of the skin are considered to be immature dendritic cells (DCs). They are CSF-1 responsive,41,42 but in the absence of CSF-1 in the Csf1op/Csf1op mouse their numbers are unaffected.43,44 EGFP was detected in the Langerhans cells either in section (Figure 6J, arrow) or in en face view in epidermal sheets (Figure 6K). In the spleen, the EGFP marker was detectable at a low level in cells forming a reticular network within the white pulp, consistent with expression in interdigitating DCs.45 Similarly, in the mesenteric lymph node, EGFP was expressed at high levels on cells within the medullary cords, where F4/80 is also expressed,27 but also on presumed interdigitating cells within lymphoid follicles (not shown). The myeloid/DC component of thymus is complex.46 EGFP was very abundant in large stellate cells especially concentrated around the cortical medullary junction (Figure 6L). In the cortex, there was also a network of ramified EGFP expression that may correspond to thymic DCs.
Transcriptional regulatory elements of the c-fms gene The c-fms gene is expressed in trophoblasts and macrophages in both mice and humans. The first part of this study showed that the underlying mechanisms are different. Whereas the human gene reportedly utilizes a promoter in the 3' end of the upstream PDGFR- to direct expression in trophoblasts,5,11 the
mouse gene clearly uses multiple transcription start sites within the
500-bp flanking exon 2. This region is actually conserved between mouse
and human, and contains elements such as AP-1 sites that have
been implicated in trophoblast-specific transcription. These
proximal sequences might contribute to activation of the upstream
promoter, or splicing, in the human gene expressed in trophoblasts. The
7.2-kb fms promoter could be utilized to direct expression
of a trophoblast-specific transgene for functional/immunologic studies.
Because the upstream promoter is not used in macrophages and is not
required for promoter activity in transient
transfections,3 we may be able to selectively delete the
trophoblast control elements to eliminate trophoblast expression while
retaining macrophage promoter activity.
Analysis of multiple independent transgenic lines confirmed that the activity of the 7.2-kb promoter construct was largely, or completely, abolished by either deletion of the first intron or elimination of the FIRE sequence. As observed in stably transfected cell lines,4 there was detectable residual activity in macrophages with the promoter alone, in keeping with reports of successful use of the corresponding human promoter region to drive low-level expression of a transgene.47 Elimination of FIRE in an intron-containing construct, on the other hand, abolished all activity.4 We have proposed that FIRE acts to overcome a block to transcription elongation in the first intron4; these findings extend the evidence favoring this model to a transgene in vivo. Based on the constitutive activity in a wide range of mouse tumor cell lines,3,13 we might have anticipated that the 3.5-kb c-fms promoter would generate ectopic expression in transgenic mice. In contrast to the data with tumor cells, the 3.5-kb promoter was very weakly active in untransformed 3T3 fibroblasts.12 The findings with the transgenic mice support the view that constitutive fms promoter activity in tumor lines reflects their malignancy.13 Interestingly, deletion of the trophoblast promoter region newly identified herein abolished c-fms promoter function in nonmacrophage tumor cells but not in RAW264 cells.13 Definition of the mononuclear phagocyte system The mononuclear phagocyte system was defined as a family of cells arising from bone marrow progenitors, circulating as monocytes and entering the tissues where they form the resident macrophage population.48 In the mouse, F4/80, which detects a member of the EGF-TM7 family of surface receptors,49 has been most widely studied as a marker of tissue macrophages.26-32 The F4/80 immunoreactivity defined a population of cells in almost every organ of the body that shared characteristic locations and stellate morphology and included most known macrophagelike cells. However, F4/80 is not detectable on progenitor cells, and is difficult to detect on blood monocytes and several tissue macrophage populations, notably in lymphoid tissues, intestine, and lung.27,30 The pattern of the c-fms-EGFP transgene was indistinguishable from published pictures of F4/80 in many locations. In addition to the well-documented macrophage populations of the liver and the red pulp of spleen, EGFP, like F4/80, was expressed in the large peri-epithelial macrophagelike cell populations of the kidney and gastrointestinal tract26,30 as well as the sinusoidal and interstitial populations of endocrine organs.31 For this reason, we have dubbed fms-EGFP mouse lines the MacGreen mouse, recognizing that there will be many applications of such animals in the study of innate immunity and experimental pathology.Tissue macrophages are thought to adapt to local environments to perform tissue-specific functions. For example, the interstitial macrophages of the testis shown clearly in Figure 6H are thought to regulate testosterone production37 and the CSF-1-deficient mouse has reproductive defects when they are reduced or absent.50 The transgene marker can be applied conveniently for purification of macrophages from each of these sites for phenotypic analysis. Figures 5 and 6 show the examples using bone marrow progenitor cells, lung interstitial macrophages, intestinal lamina propria, and splenic macrophages. Unlike F4/80, which appears only after the liver becomes the major hematopoietic site,21 the c-fms-EGFP transgene was expressed in phagocytelike cells throughout development. The expression in the embryo was indistinguishable from our earlier description of the location of c-fms mRNA.7,8 We have been able to sort fluorescent cells from enzymatically digested embryos (not shown), providing the opportunity to delineate further the ways that they differ from macrophages in adult tissues. Given the central function of CSF-1 in macrophage differentiation, and the lack of macrophages in CSF-1-deficient Csf1op/Csf1op mice44 or in the recently characterized c-fms knock out,51 the CSF-1R is the most obvious definitive marker for the mononuclear phagocyte lineage. We infer that the expression of c-fms-EGFP correlates with expression of a functional CSF-1 receptor. Colocalization of EGFP and surface CSF-1R in bone marrow cells supported this view (Figure 5). There was a population of cells that were EGFP+ but lacked detectable surface CSF-1R, but these cells expressed myeloid markers, CD11b, and to a lesser extent, F4/80 (Figure 5). CSF-1R is acutely down-modulated from the cell surface by a range of stimuli including ligand,52,53 whereas EGFP is stable. Hence, it is not surprising that the correlation is imperfect in progenitor cells and proliferating myeloid precursors. In peripheral blood, there was no detectable expression of EGFP in mature granulocytes. In the mononuclear cell fraction, and in peritoneal exudates, there was a very good correlation between EGFP and expression of F4/80 and CD11b, indicating that the transgene is restricted to monocyte macrophages and is absent from T or B lymphocytes. CSF-1-independent populations of mononuclear phagocytes The widespread expression of the transgene on all known F4/80-positive macrophage populations, including Langerhans cells, in MacGreen mice raises the issue of the existence of CSF-1-independent macrophage populations, and the relationship between macrophages and DCs. In the CSF-1-deficient Csf1op/Csf1op mouse, the majority of F4/80-positive macrophage populations are depleted, but not absent, whereas others, notably those of lymphoid organs and the skin, are unaffected.44 Even the CSF-1-dependent bone cells of the Csf1op/Csf1op mouse increase in number with age,54 a phenomenon that has been attributed to vascular endothelial growth factor A,55 which can signal macrophages through the fms-like receptor tyrosine kinase flt-1.56 Lymphoid organ T-cell area DC and skin LC numbers were also not substantially altered in Csf1op/Csf1op mice.43,44,57 This observation does not imply that subpopulations of F4/80-positive cells in vivo are CSF-1-unresponsive, or have differentiated from a separate progenitor cell from CSF-1-responsive monocyte-macrophages. There is, in fact, evidence that LCs and mature DCs respond to CSF-1 and that the factor can influence divergence of the macrophage/DC functional fate.42 We may infer that other factors in particular tissue locations can substitute for CSF-1. Ongoing studies in our laboratory will address the relationship between EGFP expression and other DC markers.Conclusion We have identified the elements within the c-fms gene required for reproducible expression of a reporter gene in cells of the mononuclear phagocyte lineage. The c-fms-EGFP transgene will be a useful marker for further studies of the biology of the cells in this system.
Thanks to Ms Elizabeth Williams for help in all aspects of the production and analysis of the transgenic mice in this project.
Submitted February 21, 2002; accepted September 4, 2002.
Prepublished online as Blood First Edition Paper, September 12, 2002; DOI 10.1182/blood-2002-02-0569.
Supported by grants from the Australian National Health and Medical Research Council to D.A.H. R.T.S. and D.O. are recipients of the Australian International Postgraduate Research Scholarship and University of Queensland International Postgraduate Research Scholarship.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: David A. Hume, Institute for Molecular Bioscience, University of Queensland, Brisbane, Q4072, Australia; e-mail: d.hume{at}imb.uq.edu.au.
1. Stanley ER, Berg KL, Einstein DB, et al. Biology and action of colony-stimulating factor-1. Mol Reprod Dev. 1997;46:4-10[CrossRef][Medline] [Order article via Infotrieve]. 2. Xie Y, Chen C, Hume DA. Transcriptional regulation of c-fms gene expression. Cell Biochem Biophys. 2001;34:1-16[Medline] [Order article via Infotrieve].
3.
Yue X, Favot P, Dunn TL, Cassady AI, Hume DA.
Expression of mRNA encoding the macrophage colony-stimulating factor receptor (c-fms) is controlled by a constitutive promoter and tissue-specific transcription elongation.
Mol Cell Biol.
1993;13:3191-3201 4. Himes SR, Tagoh H, Goonetilleke N, et al. A highly conserved c-fms gene intronic element controls macrophage-specific and regulated expression. J Leukoc Biol. 2001;70:812820.
5.
Roberts WM, Shapiro LH, Ashmun RA, Look AT.
Transcription of the human colony-stimulating factor-1 receptor gene is regulated by separate tissue-specific promoters.
Blood.
1992;79:586-593
6.
Zhang DE, Hetherington CJ, Chen HM, Tenen DG.
The macrophage transcription factor PU.1 directs tissue-specific expression of the macrophage colony-stimulating factor receptor.
Mol Cell Biol.
1994;14:373-381
7.
Lichanska AM, Browne CM, Henkel GW, et al.
Differentiation of the mononuclear phagocyte system during mouse embryogenesis: the role of transcription factor PU.1.
Blood.
1999;94:127-138 8. Hume DA, Monkley SJ, Wainwright BJ. Detection of c-fms protooncogene in early mouse embryos by whole mount in situ hybridization indicates roles for macrophages in tissue remodelling. Br J Haematol. 1995;90:939-942[Medline] [Order article via Infotrieve].
9.
Arceci RJ, Shanahan F, Stanley ER, Pollard JW.
Temporal expression and location of colony-stimulating factor 1 (CSF-1) and its receptor in the female reproductive tract are consistent with CSF-1-regulated placental development.
Proc Natl Acad Sci U S A.
1989;86:8818-8822 10. Regenstreif LJ, Rossant J. Expression of the c-fms proto-oncogene and of the cytokine, CSF-1, during mouse embryogenesis. Dev Biol. 1989;133:284-294[CrossRef][Medline] [Order article via Infotrieve].
11.
Visvader J, Verma IM.
Differential transcription of exon 1 of the human c-fms gene in placental trophoblasts and monocytes.
Mol Cell Biol.
1989;9:1336-1341
12.
Reddy MA, Yang BS, Yue X, et al.
Opposing actions of c-ets/PU.1 and c-myb protooncogene products in regulating the macrophage-specific promoters of the human and mouse colony-stimulating factor-1 receptor (c-fms) genes.
J Exp Med.
1994;180:2309-2319 13. Favot P, Yue X, Hume DA. Regulation of the c-fms promoter in murine tumour cell lines. Oncogene. 1995;11:1371-1381[Medline] [Order article via Infotrieve].
14.
Tanaka S, Kunath T, Hadjantonakis AK, Nagy A, Rossant J.
Promotion of trophoblast stem cell proliferation by FGF4.
Science.
1998;282:2072-2075 15. Morgan C, Pollard JW, Stanley ER. Isolation and characterization of a cloned growth factor dependent macrophage cell line, BAC1.2F5. J Cell Physiol. 1987;130:420-427[CrossRef][Medline] [Order article via Infotrieve]. 16. Stacey KJ, Fowles LF, Colman MS, Ostrowski MC, Hume DA. Regulation of urokinase-type plasminogen activator gene transcription by macrophage colony-stimulating factor. Mol Cell Biol. 1995;15:3430-3441[Abstract]. 17. Pavli P, Woodhams CE, Doe WF, Hume DA. Isolation and characterization of antigen-presenting dendritic cells from the mouse intestinal lamina propria. Immunology. 1990;70:40-47[Medline] [Order article via Infotrieve]. 18. Albieri A, Bevilacqua E. Induction of erythrophagocytic activity in cultured mouse trophoblast cells by phorbol myristate acetate and all-trans-retinal. Placenta. 1996;17:507-512[CrossRef][Medline] [Order article via Infotrieve]. 19. Sudo T, Nishikawa S, Ogawa M, et al. Functional hierarchy of c-kit and c-fms in intramarrow production of CFU-M. Oncogene. 1995;11:2469-2476[Medline] [Order article via Infotrieve].
20.
Passey RJ, Williams E, Lichanska AM, et al.
A null mutation in the inflammation-associated S100 protein S100A8 causes early resorption of the mouse embryo.
J Immunol.
1999;163:2209-2216 21. Lichanska AM, Hume DA. Origins and functions of phagocytes in the embryo. Exp Hematol. 2000;28:601-611[CrossRef][Medline] [Order article via Infotrieve]. 22. Dzierzak E, Medvinsky A, de Bruijn M. Qualitative and quantitative aspects of haematopoietic cell development in the mammalian embryo. Immunol Today. 1998;19:228-236[CrossRef][Medline] [Order article via Infotrieve].
23.
Anderson KL, Smith KA, Conners K, McKercher SR, Maki RA, Torbett BE.
Myeloid development is selectively disrupted in PU.1 null mice.
Blood.
1998;91:3702-3710
24.
Ross IL, Yue X, Ostrowski MC, Hume DA.
Interaction between PU.1 and another Ets family transcription factor promotes macrophage-specific Basal transcription initiation.
J Biol Chem.
1998;273:6662-6669 25. Flotte TJ, Springer TA, Thorbecke GJ. Dendritic cell and macrophage staining by monoclonal antibodies in tissue sections and epidermal sheets. Am J Pathol. 1983;111:112-124[Abstract].
26.
Hume DA, Gordon S.
Mononuclear phagocyte system of the mouse defined by immunohistochemical localization of antigen F4/80: identification of resident macrophages in renal medullary and cortical interstitium and the juxtaglomerular complex.
J Exp Med.
1983;157:1704-1709
27.
Hume DA, Robinson AP, MacPherson GG, Gordon S.
The mononuclear phagocyte system of the mouse defined by immunohistochemical localization of antigen F4/80: relationship between macrophages, Langerhans cells, reticular cells, and dendritic cells in lymphoid and hematopoietic organs.
J Exp Med.
1983;158:1522-1536
28.
Hume DA, Perry VH, Gordon S.
Immunohistochemical localization of a macrophage-specific antigen in developing mouse retina: phagocytosis of dying neurons and differentiation of microglial cells to form a regular array in the plexiform layers.
J Cell Biol.
1983;97:253-257 29. Hume DA, Loutit JF, Gordon S. The mononuclear phagocyte system of the mouse defined by immunohistochemical localization of antigen F4/80: macrophages of bone and associated connective tissue. J Cell Sci. 1984;66:189-194[Abstract]. 30. Hume DA, Perry VH, Gordon S. The mononuclear phagocyte system of the mouse defined by immunohistochemical localisation of antigen F4/80: macrophages associated with epithelia. Anat Rec. 1984;210:503-512[CrossRef][Medline] [Order article via Infotrieve].
31.
Hume DA, Halpin D, Charlton H, Gordon S.
The mononuclear phagocyte system of the mouse defined by immunohistochemical localization of antigen F4/80: macrophages of endocrine organs.
Proc Natl Acad Sci U S A.
1984;81:4174-4177 32. Hume DA, Gordon S. The mononuclear phagocytes system of the mouse defined by immunohistochemical localisation of antigen F4/80. In: van Furth R, ed. Mononuclear Phagocytes: Characteristic, Physiology and Function. Boston, MA: Martinus Nijhoff; 1985:9-17. 33. Holt PG, Warner LA, Papadimitriou JM. Alveolar macrophages: functional heterogeneity within macrophage populations from rat lung. Aust J Exp Biol Med Sci. 1982;60:607-618[Medline] [Order article via Infotrieve]. 34. Michaelson MD, Bieri PL, Mehler MF, et al. CSF-1 deficiency in mice results in abnormal brain development. Development. 1996;122:2661-2672[Abstract]. 35. Tkachuk M, Gisler RH. The promoter of macrophage colony-stimulating factor receptor is active in astrocytes. Neurosci Lett. 1997;225:121-125[CrossRef][Medline] [Order article via Infotrieve]. 36. Perry VH, Hume DA, Gordon S. Immunohistochemical localization of macrophages and microglia in the adult and developing mouse brain. Neuroscience. 1985;15:313-326[CrossRef][Medline] [Order article via Infotrieve]. 37. Cohen PE, Nishimura K, Zhu L, Pollard JW. Macrophages: important accessory cells for reproductive function. J Leukoc Biol. 1999;66:765-772[Abstract]. 38. Edwards M, Sarma U, Flanagan AM. Macrophage colony-stimulating factor increases bone resorption by osteoclasts disaggregated from human fetal long bones. Bone. 1998;22:325-329[Medline] [Order article via Infotrieve]. 39. Kawakami M, Kuroda S, Yamashita K, Yoshida CA, Nakagawa K, Takada K. Expression of CSF-1 receptor on TRAP-positive multinuclear cells around the erupting molars in rats. J Craniofac Genet Dev Biol. 1999;19:213-220[Medline] [Order article via Infotrieve]. 40. van Furth R. Production and migration of monocytes and kinetics of macrophages. In: van Furth R, ed. Mononuclear Phagocytes: Biology of Monocytes and Macrophages. The Netherlands: Kluwer Academic; 1992:3-12. 41. Koyama Y, Marunouchi T. Macrophage colony-stimulating factor (M-CSF) inhibits the decrease in the amount of rRNA and IA beta mRNA in cultured epidermal Langerhans cells of the mouse. J Dermatol. 1996;23:73-82[Medline] [Order article via Infotrieve]. 42. Xu S, Ariizumi K, Edelbaum D, Bergstresser PR, Takashima A. Cytokine-dependent regulation of growth and maturation in murine epidermal dendritic cell lines. Eur J Immunol. 1995;25:1018-1024[Medline] [Order article via Infotrieve]. 43. Usuda H, Naito M, Umeda S, Takahashi K, Shultz LD. Ultrastructure of macrophages and dendritic cells in osteopetrosis (op) mutant mice lacking macrophage colony-stimulating factor (M-CSF/CSF-1) activity. J Submicrosc Cytol Pathol. 1994;26:111-119[Medline] [Order article via Infotrieve]. 44. Cecchini MG, Dominguez MG, Mocci S, et al. Role of colony stimulating factor-1 in the establishment and regulation of tissue macrophages during postnatal development of the mouse. Development. 1994;120:1357-1372[Abstract]. 45. Steinman RM, Pack M, Inaba K. Dendritic cells in the T-cell areas of lymphoid organs. Immunol Rev. 1997;156:25-37[CrossRef][Medline] [Order article via Infotrieve]. 46. Shortman K, Vremec D, Corcoran LM, Georgopoulos K, Lucas K, Wu L. The linkage between T-cell and dendritic cell development in the mouse thymus. Immunol Rev. 1998;165:39-46[CrossRef][Medline] [Order article via Infotrieve]. 47. Jin DI, Jameson SB, Reddy MA, Schenkman D, Ostrowski MC. Alterations in differentiation and behavior of monocytic phagocytes in transgenic mice that express dominant suppressors of ras signaling. Mol Cell Biol. 1995;15:693-703[Abstract]. 48. van Furth R, Cohn ZA, Hirsch JG, Humphrey JH, Spector WG, Langevoort HL. The mononuclear phagocyte system: a new classification of macrophages, monocytes, and their precursor cells. Bull World Health Organ. 1972;46:845-852[Medline] [Order article via Infotrieve]. 49. McKnight AJ, Gordon S. EGF-TM7: a novel subfamily of seven-transmembrane-region leukocyte cell-surface molecules. Immunol Today. 1996;17:283-287[CrossRef][Medline] [Order article via Infotrieve]. 50. Cohen PE, Chisholm O, Arceci RJ, Stanley ER, Pollard JW. Absence of colony-stimulating factor-1 in osteopetrotic (csfmop/csfmop) mice results in male fertility defects. Biol Reprod. 1996;55:310-317[Abstract].
51.
Dai XM, Ryan GR, Hapel AJ, et al.
Targeted disruption of the mouse colony-stimulating factor 1 receptor gene results in osteopetrosis, mononuclear phagocyte deficiency, increased primitive progenitor cell frequencies, and reproductive defects.
Blood.
2002;99:111-120
52.
Sester DP, Beasley SJ, Sweet MJ, et al.
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.
J Immunol.
1999;163:6541-6550 53. Fowles LF, Stacey KJ, Marks D, Hamilton JA, Hume DA. Regulation of urokinase plasminogen activator gene transcription in the RAW264 murine macrophage cell line by macrophage colony-stimulating factor (CSF-1) is dependent upon the level of cell-surface receptor. Biochem J. 2000;347:313-320. 54. Pollard JW, Stanley ER. Pleiotropic roles for CSF-1 in development defined by mouse mutation, osteopetrotic. Ad Develop Biochem. 1996;4:153-193.
55.
Niida S, Kaku M, Amano H, et al.
Vascular endothelial growth factor can substitute for macrophage colony-stimulating factor in the support of osteoclastic bone resorption.
J Exp Med.
1999;190:293-298
56.
Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z.
Vascular endothelial growth factor (VEGF) and its receptors.
Faseb J.
1999;13:9-22 57. Witmer-Pack MD, Hughes DA, Schuler G, et al. Identification of macrophages and dendritic cells in the osteopetrotic (op/op) mouse. J Cell Sci. 1993;104:1021-1029[Abstract].
© 2003 by The American Society of Hematology.
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  C. Auffray, D. K. Fogg, E. Narni-Mancinelli, B. Senechal, C. Trouillet, N. Saederup, J. Leemput, K. Bigot, L. Campisi, M. Abitbol, et al. CX3CR1+ CD115+ CD135+ common macrophage/DC precursors and the role of CX3CR1 in their response to inflammation J. Exp. Med., March 16, 2009; 206(3): 595 - 606. [Abstract] [Full Text] [PDF]
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 Q. Sun, P. Yue, J. A. Deiuliis, C. N. Lumeng, T. Kampfrath, M. B. Mikolaj, Y. Cai, M. C. Ostrowski, B. Lu, S. Parthasarathy, et al. Ambient Air Pollution Exaggerates Adipose Inflammation and Insulin Resistance in a Mouse Model of Diet-Induced Obesity Circulation, February 3, 2009; 119(4): 538 - 546. [Abstract] [Full Text] [PDF]
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P. Qu, H. Du, Y. Li, and C. Yan Myeloid-Specific Expression of Api6/AIM/Sp{alpha} Induces Systemic Inflammation and Adenocarcinoma in the Lung J. Immunol., February 1, 2009; 182(3): 1648 - 1659. [Abstract] [Full Text] [PDF]
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M. K. Chang, L.-J. Raggatt, K. A. Alexander, J. S. Kuliwaba, N. L. Fazzalari, K. Schroder, E. R. Maylin, V. M. Ripoll, D. A. Hume, and A. R. Pettit Osteal Tissue Macrophages Are Intercalated throughout Human and Mouse Bone Lining Tissues and Regulate Osteoblast Function In Vitro and In Vivo J. Immunol., July 15, 2008; 181(2): 1232 - 1244. [Abstract] [Full Text] [PDF]
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D. A. Hume, T. Sasmono, S. R. Himes, S. M. Sharma, A. Bronisz, M. Constantin, M. C. Ostrowski, and I. L. Ross The Ewing Sarcoma Protein (EWS) Binds Directly to the Proximal Elements of the Macrophage-Specific Promoter of the CSF-1 Receptor (csf1r) Gene J. Immunol., May 15, 2008; 180(10): 6733 - 6742. [Abstract] [Full Text] [PDF]
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 D. A. Ovchinnikov, W. J. M. van Zuylen, C. E. E. DeBats, K. A. Alexander, S. Kellie, and D. A. Hume Expression of Gal4-dependent transgenes in cells of the mononuclear phagocyte system labeled with enhanced cyan fluorescent protein using Csf1r-Gal4VP16/UAS-ECFP double-transgenic mice J. Leukoc. Biol., February 1, 2008; 83(2): 430 - 433. [Abstract] [Full Text] [PDF]
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K. Weigelt, W. Ernst, Y. Walczak, S. Ebert, T. Loenhardt, M. Klug, M. Rehli, B. H. F. Weber, and T. Langmann Dap12 expression in activated microglia from retinoschisin-deficient retina and its PU.1-dependent promoter regulation J. Leukoc. Biol., December 1, 2007; 82(6): 1564 - 1574. [Abstract] [Full Text] [PDF]
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 M. Buck and M. Chojkier C/EBP phosphorylation rescues macrophage dysfunction and apoptosis induced by anthrax lethal toxin Am J Physiol Cell Physiol, December 1, 2007; 293(6): C1788 - C1796. [Abstract] [Full Text] [PDF]
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R. T. Sasmono, A. Ehrnsperger, S. L. Cronau, T. Ravasi, R. Kandane, M. J. Hickey, A. D. Cook, S. R. Himes, J. A. Hamilton, and D. A. Hume Mouse neutrophilic granulocytes express mRNA encoding the macrophage colony-stimulating factor receptor (CSF-1R) as well as many other macrophage-specific transcripts and can transdifferentiate into macrophages in vitro in response to CSF-1 J. Leukoc. Biol., July 1, 2007; 82(1): 111 - 123. [Abstract] [Full Text] [PDF]
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X. Z. Shen, P. Li, D. Weiss, S. Fuchs, H. D. Xiao, J. A. Adams, I. R. Williams, M. R. Capecchi, W. R. Taylor, and K. E. Bernstein Mice with Enhanced Macrophage Angiotensin-Converting Enzyme Are Resistant to Melanoma Am. J. Pathol., June 1, 2007; 170(6): 2122 - 2134. [Abstract] [Full Text] [PDF]
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V. M. Ripoll, K. M. Irvine, T. Ravasi, M. J. Sweet, and D. A. Hume Gpnmb Is Induced in Macrophages by IFN-{gamma} and Lipopolysaccharide and Acts as a Feedback Regulator of Proinflammatory Responses J. Immunol., May 15, 2007; 178(10): 6557 - 6566. [Abstract] [Full Text] [PDF]
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 J. B. Wyckoff, Y. Wang, E. Y. Lin, J.-f. Li, S. Goswami, E. R. Stanley, J. E. Segall, J. W. Pollard, and J. Condeelis Direct Visualization of Macrophage-Assisted Tumor Cell Intravasation in Mammary Tumors Cancer Res., March 15, 2007; 67(6): 2649 - 2656. [Abstract] [Full Text] [PDF]
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 H. Krysinska, M. Hoogenkamp, R. Ingram, N. Wilson, H. Tagoh, P. Laslo, H. Singh, and C. Bonifer A Two-Step, PU.1-Dependent Mechanism for Developmentally Regulated Chromatin Remodeling and Transcription of the c-fms Gene Mol. Cell. Biol., February 1, 2007; 27(3): 878 - 887. [Abstract] [Full Text] [PDF]
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S. Wei, X.-M. Dai, and E. R. Stanley Transgenic expression of CSF-1 in CSF-1 receptor-expressing cells leads to macrophage activation, osteoporosis, and early death J. Leukoc. Biol., December 1, 2006; 80(6): 1445 - 1453. [Abstract] [Full Text] [PDF]
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 K. Inoue, M. Kobayashi, K. Yano, M. Miura, A. Izumi, C. Mataki, T. Doi, T. Hamakubo, P. C. Reid, D. A. Hume, et al. Histone Deacetylase Inhibitor Reduces Monocyte Adhesion to Endothelium Through the Suppression of Vascular Cell Adhesion Molecule-1 Expression Arterioscler. Thromb. Vasc. Biol., December 1, 2006; 26(12): 2652 - 2659. [Abstract] [Full Text] [PDF]
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E. Y. Lin, J.-F. Li, L. Gnatovskiy, Y. Deng, L. Zhu, D. A. Grzesik, H. Qian, X.-n. Xue, and J. W. Pollard Macrophages Regulate the Angiogenic Switch in a Mouse Model of Breast Cancer Cancer Res., December 1, 2006; 66(23): 11238 - 11246. [Abstract] [Full Text] [PDF]
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 M. E. Rothenberg, M. P. Doepker, I. P. Lewkowich, M. G. Chiaramonte, K. F. Stringer, F. D. Finkelman, C. L. MacLeod, L. G. Ellies, and N. Zimmermann Cationic amino acid transporter 2 regulates inflammatory homeostasis in the lung PNAS, October 3, 2006; 103(40): 14895 - 14900. [Abstract] [Full Text] [PDF]
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J. E. Qualls, A. M. Kaplan, N. van Rooijen, and D. A. Cohen Suppression of experimental colitis by intestinal mononuclear phagocytes J. Leukoc. Biol., October 1, 2006; 80(4): 802 - 815. [Abstract] [Full Text] [PDF]
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M.-H. Jang, D. M. Herber, X. Jiang, S. Nandi, X.-M. Dai, G. Zeller, E. R. Stanley, and V. R. Kelley Distinct In Vivo Roles of Colony-Stimulating Factor-1 Isoforms in Renal Inflammation J. Immunol., September 15, 2006; 177(6): 4055 - 4063. [Abstract] [Full Text] [PDF]
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 G. Lee, A. Lo, S. A. Short, T. J. Mankelow, F. Spring, S. F. Parsons, K. Yazdanbakhsh, N. Mohandas, D. J. Anstee, and J. A. Chasis Targeted gene deletion demonstrates that the cell adhesion molecule ICAM-4 is critical for erythroblastic island formation Blood, September 15, 2006; 108(6): 2064 - 2071. [Abstract] [Full Text] [PDF]
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C. Yan, X. Lian, Y. Li, Y. Dai, A. White, Y. Qin, H. Li, D. A. Hume, and H. Du Macrophage-Specific Expression of Human Lysosomal Acid Lipase Corrects Inflammation and Pathogenic Phenotypes in lal-/- Mice Am. J. Pathol., September 1, 2006; 169(3): 916 - 926. [Abstract] [Full Text] [PDF]
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D. A. Hume Comment on "CCR7 Is Critically Important for Migration of Dendritic Cells in Intestinal Lamina Propria to Mesenteric Lymph Nodes" J. Immunol., August 15, 2006; 177(4): 2035 - 2035. [Full Text] [PDF]
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 G. A. Challen, I. Bertoncello, J. A. Deane, S. D. Ricardo, and M. H. Little Kidney Side Population Reveals Multilineage Potential and Renal Functional Capacity but also Cellular Heterogeneity J. Am. Soc. Nephrol., July 1, 2006; 17(7): 1896 - 1912. [Abstract] [Full Text] [PDF]
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 L Banaei-Bouchareb, M Peuchmaur, P Czernichow, and M Polak A transient microenvironment loaded mainly with macrophages in the early developing human pancreas. J. Endocrinol., March 1, 2006; 188(3): 467 - 480. [Abstract] [Full Text] [PDF]
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S. R. Himes, D. P. Sester, T. Ravasi, S. L. Cronau, T. Sasmono, and D. A. Hume The JNK Are Important for Development and Survival of Macrophages J. Immunol., February 15, 2006; 176(4): 2219 - 2228. [Abstract] [Full Text] [PDF]
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M. Anghelina, P. Krishnan, L. Moldovan, and N. I. Moldovan Monocytes/Macrophages Cooperate with Progenitor Cells during Neovascularization and Tissue Repair: Conversion of Cell Columns into Fibrovascular Bundles Am. J. Pathol., February 1, 2006; 168(2): 529 - 541. [Abstract] [Full Text] [PDF]
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J. Y. Bertrand, A. Jalil, M. Klaine, S. Jung, A. Cumano, and I. Godin Three pathways to mature macrophages in the early mouse yolk sac Blood, November 1, 2005; 106(9): 3004 - 3011. [Abstract] [Full Text] [PDF]
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K. P. A. MacDonald, V. Rowe, H. M. Bofinger, R. Thomas, T. Sasmono, D. A. Hume, and G. R. Hill The Colony-Stimulating Factor 1 Receptor Is Expressed on Dendritic Cells during Differentiation and Regulates Their Expansion J. Immunol., August 1, 2005; 175(3): 1399 - 1405. [Abstract] [Full Text] [PDF]
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 L. P. Ganesan, G. Wei, R. A. Pengal, L. Moldovan, N. Moldovan, M. C. Ostrowski, and S. Tridandapani The Serine/Threonine Kinase Akt Promotes Fc{gamma} Receptor-mediated Phagocytosis in Murine Macrophages through the Activation of p70S6 Kinase J. Biol. Chem., December 24, 2004; 279(52): 54416 - 54425. [Abstract] [Full Text] [PDF]
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 A. Schober, A. Zernecke, E. A. Liehn, P. von Hundelshausen, S. Knarren, W. A. Kuziel, and C. Weber Crucial Role of the CCL2/CCR2 Axis in Neointimal Hyperplasia After Arterial Injury in Hyperlipidemic Mice Involves Early Monocyte Recruitment and CCL2 Presentation on Platelets Circ. Res., November 26, 2004; 95(11): 1125 - 1133. [Abstract] [Full Text] [PDF]
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 A Amarante-Paffaro, G S Queiroz, S T Correa, B Spira, and E Bevilacqua Phagocytosis as a potential mechanism for microbial defense of mouse placental trophoblast cells Reproduction, August 1, 2004; 128(2): 207 - 218. [Abstract] [Full Text] [PDF]
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L. Banaei-Bouchareb, V. Gouon-Evans, D. Samara-Boustani, M. C. Castellotti, P. Czernichow, J. W. Pollard, and M. Polak Insulin cell mass is altered in Csf1op/Csf1op macrophage-deficient mice J. Leukoc. Biol., August 1, 2004; 76(2): 359 - 367. [Abstract] [Full Text] [PDF]
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S. H. Burnett, E. J. Kershen, J. Zhang, L. Zeng, S. C. Straley, A. M. Kaplan, and D. A. Cohen Conditional macrophage ablation in transgenic mice expressing a Fas-based suicide gene J. Leukoc. Biol., April 1, 2004; 75(4): 612 - 623. [Abstract] [Full Text] [PDF]
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B. J. Jenkins, D. Grail, M. Inglese, C. Quilici, S. Bozinovski, P. Wong, and M. Ernst Imbalanced gp130-Dependent Signaling in Macrophages Alters Macrophage Colony-Stimulating Factor Responsiveness via Regulation of c-fms Expression Mol. Cell. Biol., February 15, 2004; 24(4): 1453 - 1463. [Abstract] [Full Text] [PDF]
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 G. A. Follows, H. Tagoh, P. Lefevre, G. J. Morgan, and C. Bonifer Differential transcription factor occupancy but evolutionarily conserved chromatin features at the human and mouse M-CSF (CSF-1) receptor loci Nucleic Acids Res., October 15, 2003; 31(20): 5805 - 5816. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
5') primers located in exon 2. Primer sequences for
5'RACE experiments are described in Table
Zap
vector (Clontech, Palo Alto, CA). Average library insert size was
1.5 kb and its complexity was 1.5 × 106 independent
clones. This library was probed with a
[32P]-dCTP-labeled full-length c-fms cDNA in
the first round followed by a c-fms exon 2 probe in the
second round. The 5' ends of the selected inserts were sequenced.
-modification medium (Invitrogen) supplemented with 10% FCS, 20 U/mL penicillin, and 20 µg/mL streptomycin, in the presence or
absence of 100 U/mL CSF-1 (Chiron).
