Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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Originally published online as doi:10.1189/jlb.0904515 on May 13, 2005

Published online before print May 13, 2005
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(Journal of Leukocyte Biology. 2005;78:481-490.)
© 2005 by Society for Leukocyte Biology

Identification of CCR2, flotillin, and gp49B genes as new G-CSF targets during neutrophilic differentiation

Satoshi Iida*, Takahide Kohro{dagger}, Tatsuhiko Kodama{dagger}, Shigekazu Nagata*,{ddagger},§ and Rikiro Fukunaga*,{ddagger},§,1

* Department of Genetics, B-3, Graduate School of Medicine, and
{ddagger} Graduate School of Frontier Biosciences, Osaka University, Japan;
§ Solution-Oriented Research for Science and Technology (SORST), Japan Science and Technology Agency, Osaka, Japan; and
{dagger} Laboratory of System Biology and Medicine, Research Center for Advanced Science and Technology, University of Tokyo, Japan

1 Correspondence: Department of Genetics, B-3, Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: fukunaga{at}genetic.med.osaka-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Granulocyte colony-stimulating factor (G-CSF) is a cytokine that stimulates myeloid progenitor cells to proliferate and differentiate into neutrophilic granulocytes. To identify genes induced by G-CSF during neutrophil differentiation, interleukin-3-dependent murine myeloid precursor FDC-P1 cells expressing the G-CSF receptor were stimulated with G-CSF, and the gene expression profile was characterized by DNA microarray analysis. In addition to known signal transducer and activator of transcription-3 target genes, such as suppressor of cytokine signaling-3 (SOCS3), JunB, and p19INK4D, we newly identified several G-CSF targets, including genes for the CC chemokine receptor-2 (CCR2), raft proteins flotillin-1 and flotillin-2, and immunoglobulin-like receptor gp49B. Real-time, quantitative polymerase chain reaction analyses revealed that the expression of these genes was induced in various myeloid cell lines by G-CSF. Furthermore, when HoxA9-immortalized bone marrow progenitors were induced by G-CSF to differentiate into mature neutrophils, all of these genes were strongly activated. These genes could be categorized into three groups based on their time-course of expression: immediate-early (~20 min, SOCS3), mid-early (2–4 h, flotillin-1/2 and gp49B), and late (>12 h, CCR2). This suggests that different transcriptional mechanisms are involved in the regulation of these genes. We show that bone marrow neutrophils express functional CCR2, which suggest that CC chemokines may play previously unknown roles in neutrophil activation and chemotaxis.

Key Words: DNA microarray • GM-CSF • M-CSF • SOCS • HoxA9


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neutrophilic granulocytes (neutrophils) constitute the major population of white blood cells and play a critical role in innate immunity by protecting the host against infection by ingesting and killing invading microorganisms. Neutrophils are produced in the bone marrow, where myeloid progenitor cells derived from the hematopoietic stem cells proliferate and differentiate [1 ]. Granulocyte colony-stimulating factor (G-CSF) is a cytokine that controls granulopoiesis by regulating the proliferation and differentiation of myeloid progenitor cells into mature neutrophils. Studies of knockout mice deficient in G-CSF or the G-CSF receptor (G-CSFR) have demonstrated the importance of G-CSF as a major regulator of steady-state granulopoiesis and of emergency granulopoiesis during infection [2 , 3 ]. Upon binding to G-CSFR, expressed on myeloid progenitors, G-CSF stimulates multiple intracellular signaling pathways via the activation of the Janus family kinases, JAK1 and JAK2, which phosphorylate tyrosine residues in the cytoplasmic domain of G-CSFR, and the signal transducer and activator of transcription (STAT) family proteins, such as STAT3 and STAT5 [4 5 6 ]. Tyrosine-phosphorylated STAT3 dimerizes, translocates into the nucleus, and activates various target genes, including the suppressors of cytokine signaling protein (SOCS), and the phosphotyrosine residues in the G-CSFR cytoplasmic domain recruit various proteins, including Grb2, Shc, Src homology (SH)-2-containing tyrosine phosphatase-1 (SHP-1), and SHP-2, to activate other signaling pathways, such as the Ras-mitogen-activated protein kinase cascades [6 ]. Studies using various mutants of G-CSFR or STAT3 have demonstrated that STAT3 activation by G-CSF is important for the proliferation and differentiation of neutrophilic progenitors [7 8 9 10 ], whereas other reports have suggested that STAT3 is dispensable for in vivo granulopoiesis but is instead involved in regulating the activity of terminally differentiated neutrophils [11 , 12 ].

Mature neutrophils possess specific proteins, such as myeloperoxidase (MPO), neutrophil elastase (NE), reduced nicotinamide adenine dinucleotide phosphate oxidase, and lysozyme, for their bacteriocidal activity. Numerous studies of the transcriptional regulation of these marker proteins have demonstrated that the expression of neutrophil-specific genes requires transcription factors, including CCAAT/enhancer-binding protein (C/EBP) family members, acute myeloid leukemia 1a (AML-1a), myeloid zinc finger protein-1 or -2A (MZF-1/2A), PU.1, NF-Y, and c-Myb [13 14 15 16 17 ]. Among the C/EBP members, C/EBP{alpha} is critical for G-CSFR expression [18 ] and also seems to play a predominant role in gene expression in immature neutrophils, whereas C/EBP{varepsilon} seems to regulate gene expression at the terminal stage of granulopoiesis [14 15 16 ]. Although G-CSF-induced granulopoiesis is obviously accomplished by the temporally regulated expression of specific genes under the control of these transcription factors, little is known about the expression kinetics of neutrophil-specific genes during G-CSF-induced differentiation. One way to study changes in gene expression is with the recently developed DNA microarray technique, which has been used to analyze global gene expression in a wide variety of cellular processes, including neutrophil differentiation and activation [19 20 21 ].

In the present study, we carried out a DNA microarray analysis of G-CSF-responsive myeloid precursor cells and identified several genes that were strongly induced by G-CSF stimulation. Quantitative analyses of their transcripts in myeloid cell lines and in HoxA9-immortalized bone marrow progenitors indicated that G-CSF-stimulated, immature neutrophils expressed these genes with distinct kinetics; based on their expression patterns, these genes can be categorized as immediate-early, mid-early, or late. A strong expression of the CC chemokine receptor-2 (CCR2) in G-CSF-stimulated neutrophils suggests that CC chemokines, such as monocyte chemoattractant protein-1 (MCP-1), play a role in the regulation of neutrophil functions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture, preparation of RNA, and DNA microarray analysis
Recombinant mouse interleukin-3 (rmIL-3) and granulocyte macrophage-CSF (GM-CSF) were produced in mouse C127 cells as described previously [22 ]. Recombinant human (rh)G-CSF was provided by the Chugai Pharmaceutical Company (Tokyo, Japan). IL-3-dependent mouse myeloid cell lines FDN1.1, BA3-13, LG, 32DCl3, and NFS60 [7 , 23 , 24 ] were maintained in the basic medium [RPMI-1640 medium supplemented with 10% fetal calf serum (Invitrogen, Carlsbad, CA), 50 µM 2-mercaptoethanol, 100 µg/ml kanamycin, and 100 µg/ml streptomycin] containing 45 units/ml mIL-3. For stimulation by G-CSF, cells were washed twice with the basic medium to remove IL-3 and then cultured at a density of less than 1 x 106 cells/ml in the basic medium containing 200 units/ml rhG-CSF at 37°C. Total RNA was prepared from 1 x 107 cells using the RNeasy kit with the RNase-Free DNase set (Qiagen, Valencia, CA). For the microarray analysis, total RNAs were prepared from FDN1.1 cells stimulated with G-CSF (200 u/ml) for 0, 2, 8, and 14 h. The DNA microarray analysis was carried out using the GeneChip Array murine genome U74Av2 set (Affymetrix, Santa Clara, CA) as described [25 , 26 ]. All data, including the signal intensity of each gene and fold change between them, were calculated using Microarray Analysis Suite Version 4.0 (MAS 4.0, Affymetrix). The overall signal intensity of each array was normalized so that the average would be 100. As a result of technical limitations in MAS 4.0, signal intensities of some genes were presented in negative values, even if they were actually expressed in cells. For genes whose value at time zero was negative or smaller than the estimated detection limit, the fold changes are calculated using an estimate of the minimum value for detectable transcripts. All data, including the signal intensity, fold change, and absence/presence call, can be downloaded at http://www.med.rcast.u-tokyo.ac.jp/data/fukunaga/fukunaga.xls.

Real-time polymerase chain reaction (PCR) for quantitative analysis of mRNA
For the reverse-transcribed (RT)-PCR reaction, cDNA was synthesized from DNase I-treated total RNA (0.5 µg) using an oligo-(dT) primer and Superscript III (Invitrogen) in a 10-µL reaction mixture. Real-time RT-PCR analysis was carried out using the LightCycler-FastStart DNA Master SYBR Green I kit (Roche Diagnostics, Nutley, NJ). In brief, an aliquot of the synthesized cDNA was diluted with 20 µL reaction mixture (3 mM MgCl2, 1x FastStart DNA Master SYBR Green I) containing 10 pmol each of the sense and antisense primers, and the mixture was subjected to the PCR reaction using the LightCycler system (Roche Diagnostics) under the following conditions: 15 s at 95°C, 5 s at 60°C, and 20 s at 72°C for 40 cycles. The primer sets used were: 5'-TGTGATGGTGGGAATGGGTCAG-3' and 5'-TTTGATGTCACGCACGATTTCC-3' for ß-actin, 5'-TCAGCTCCAAAAGCGAGTAC-3' and 5'-CACCAGCTTGAGTACACAGT-3' for SOCS3, 5'-AGGCCGAGTGTTTGTCCTAC-3' and 5'-GCTAACCACGCTGATACCCA-3' for flotillin-1, 5'-ATTATGACGTTGCAGCCCCG-3' and 5'-ATGGTGAAGCTGAGGATCTC-3' for flotillin-2, 5'-ACTCACAGCATCAGGCCAAT-3' and 5'-ACTGGTATCCGATGAGGATG-3' for gp49, 5'-GAGCCATACCTGTAAATGCC-3' and 5'-GAGCCCAGAATGGTAATGTG-3' for CCR2, and 5'-CCAGGAACAACATCACCATTCGCA-3' and 5'-AGAGCTTCTCCCCATTCCAT-3' for MPO. In all real-time RT-PCR experiments, cloned cDNA fragments (102–106 copies per reaction) containing the respective target region were amplified in parallel and used as standards. The real-time RT-PCR data were normalized against standards and expressed as the copy number of target mRNA per nanogram of total RNA.

Northern blot analysis
For the Northern blot analysis of SOCS family genes, FDN1.1 cells were cytokine-starved in the basic medium for 6 h, and then IL-3 or G-CSF was added to the medium. Total RNAs (5 or 10 µg) prepared at the indicated times were denatured and separated by electrophoresis on a 1% agarose gel containing 0.22 M formaldehyde. The fractionated RNA was transferred to a nylon membrane filter and subjected to Northern blot hybridization as described [24 ].

Western blotting
Cells were lysed directly in Laemmli’s sample loading buffer and heated for 30 min at 85°C and for 5 min at 95°C. Samples were separated by electrophoresis on a 10–20% gradient polyacrylamide gel (Biocraft, Tokyo, Japan) and transferred to a polyvinylidene difluoride membrane filter (Millipore, Bedford, MA). Western blot analysis was carried out using the enhanced chemiluminescence system (Perkin-Elmer, Wellesley, MA) with mouse anti-flotillin-1 or anti-flotillin-2 monoclonal antibody (mAb; BD Biosciences, San Jose, CA) and peroxidase-conjugated goat anti-mouse immunoglobulin (Ig; Dako, Copenhagen, Denmark).

Production of HoxA9 retroviral vector and immortalization of murine bone marrow progenitor cells
HoxA9-immortalized myeloid progenitor cells were established according to the protocol described previously [27 ]. The coding region of murine HoxA9 cDNA was cloned from 32DCl3 RNA by RT-PCR using the primers 5'-GGTGAATTCCACCATGGCCACCACCGGGGCCCT-3' and 5'-CCACCTCGAGTCACTCGTCTTTTGCTCGGT-3'. The cDNA was digested with EcoRI and XhoI and subcloned into the retroviral vector pMYs-IG (a kind gift from Dr. Toshio Kitamura, University of Tokyo, Japan), which was derived from the murine embryonic stem cell virus [28 ]. The resulting pMYs-HoxA9-IG plasmid was transfected into the packaging cell line Plat-E [28 ] using FuGene transfection reagent (Roche Diagnostics), and the transfected cells were cultured for 2 days to produce a helper-free retrovirus encoding HoxA9. The culture supernatant was recovered and used directly as a retrovirus stock. Bone marrow cells were isolated from the femurs and tibias of BALB/c mice, and cells enriched in myeloid progenitors were prepared by centrifugation on Ficoll-Paque Plus (Amersham Biosciences, Piscataway, NJ). One million of the progenitor cells were transferred to a 35-mm dish and incubated for 1 h in 2 ml basic medium containing 100 u/ml GM-CSF, 8 µg/ml Polybrene, and 50% of the retrovirus stock. The infected cells were diluted with 6 ml basic medium containing GM-CSF, transferred into a 100-mm dish, and cultured at 37°C under 5% CO2. After 3 days, half of the medium was removed and replaced with fresh medium containing GM-CSF. Nonadherent cells were passaged every 3 days into the same medium to keep the cell density at less than 1 x 106 cells/ml. Progenitor cells infected with a control retrovirus lacking HoxA9 did not survive for more than 4 weeks, whereas progenitor cells infected with the HoxA9 retrovirus kept proliferating stably in a GM-CSF-dependent manner. After continuous passage for more than 3 months, cells thus obtained were used as HoxA9-immortalized progenitor cells.

Wright-Giemsa staining and flow cytometry analysis
The immortalized progenitor cells were washed with basic medium and cultured for 3 days in the basic medium containing GM-CSF (100 u/ml), G-CSF (200 u/ml), or IL-3 (45 u/ml). The cells were then cytospun onto glass slides and stained with Wright-Giemsa for morphological characterization. For the analysis of surface antigens, progenitor cells stimulated with cytokines as above were stained with phycoerythrin (PE)-conjugated rat anti-mouse Gr-1 (Clone RB6-8C5, BD Biosciences) or biotinylated hamster anti-mouse F4/80 mAb (a gift from Dr. Masato Tanaka, Riken Research Center for Allergy and Immunology, Yokohama, Japan) together with streptoavidin PE (BD Biosciences). Flow cytometry analysis was carried out using a FACSCalibur (BD Biosciences).

Chemotaxis assay
Cell migration was assessed by using Transwell chambers with 3 µm pores (Costar, Corning, NY) as described [29 ]. Bone marrow cells prepared as above were suspended in assay medium [RPMI-1640 medium supplemented with 20 mM HEPES (pH 7.4) and 0.5% bovine serum albumin (BSA)]. Cell suspension (100 µl) was placed in the upper compartment (106 cells/well), and 0.6 ml phosphate-buffered saline containing 0.5% BSA and murine MCP-1 (PeproTech, Rocky Hill, NJ) was placed in the lower compartment. After incubation at 37°C for 1.5 h in 5% CO2, the plates were centrifuged at 300 gfor 5 min. The cells from the lower compartment were counted on a Coulter counter (Beckman Coulter, Fullerton, CA). In a parallel experiment, the cells migrated to the lower compartment were stained with PE-conjugated anti-mouse Gr-1 and analyzed by flow cytometry.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of G-CSF-responsive genes by DNA microarray analysis
We previously established a mouse myeloid cell line FD62M by the forced expression of G-CSFR in IL-3-dependent myeloid precursor FDC-P1 cells and showed that FD62M cells can express neutrophil-specific genes such as MPO and NE genes in response to G-CSF [23 , 24 ]. To identify other genes that are inducibly expressed during G-CSF-dependent neutrophil differentiation, we performed DNA microarray analysis using FDN1.1, a subclone of the FD62M cells. FDN1.1 cells maintained in medium containing IL-3 were washed and stimulated with G-CSF for 2, 8, and 14 h, and the total RNAs were extracted and subjected to microarray analysis using the GeneChip mouse U74Av2 set (12,488 probes). After the hybridization, data were calculated and normalized using Affymetrix MAS 4.0. We selected genes whose normalized signal intensity at 14 h was judged to be sufficiently high (>70) and then sorted these 4485 genes with the fold-change value of 14 h versus control in descending order. (The array data are available at http://www.med.rcast.u-tokyo.ac.jp/data/fukunaga/fukunaga.xls.) Genes that showed more than a fourfold increase in expression at 14 h are listed in Table 1 . The gene most strongly induced by G-CSF was SOCS3, which is known to be the direct target of STAT3 [30 31 32 ]. Other STAT3 targets, JunB [33 ] and the cyclin-dependent kinase-4 inhibitor p19INK4D [34 ], were also listed in the top ranks, indicating the reliability of this analysis. In addition to these known STAT3 targets, the genes for CCR2, flotillin-1, flotilin-2, and gp49 were identified to be strongly (>tenfold) up-regulated by G-CSF.


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  		Table 1. DNA Microarray Analysis of Genes Induced by G-CSF in FDN1.1 Cells

 
CCR2 is a seven-transmembrane-spanning CCR that binds to a group of CC chemokines including MCP-1, and it signals through heterotrimeric G-protein complexes [35 , 36 ]. It is widely known that CCR2 is expressed in monocytes, dendritic cells, natural killer (NK) cells, and T lymphocytes, but little is known about its expression in neutrophils [36 , 37 ]. The strong induction of CCR2 mRNA by G-CSF suggests that CCR2 and its ligand chemokines play roles in neutrophil function. The microarray analysis also revealed that CCR1 and CXC chemokine receptor 2 (CXCR2) genes were induced by G-CSF (7.5- and 4.1-fold changes at 14 h, respectively). Their signal intensities (59 and 48 at 14 h, respectively) were significant but lower than the threshold value we set for Table 1 . Conversely, CXCR3 and CXCR4 genes were expressed constitutively, whereas none of the CCR4, CCR5, CCR7, and CCR9 genes were expressed in FDN1.1 cells, irrespective of G-CSF stimulation (data not shown). Flotillin-1 and -2, also called reggie-2 and -1, respectively, are lipid raft-associated proteins and have been postulated to play roles in neuronal regeneration, cytokine signal transduction, and phagosome function in macrophages [38 39 40 41 ]. A previous microarray study also identified flotillin-1 as a G-CSF-responsive gene [42 ]. The induction of flotillin-1 and -2 mRNAs by G-CSF may be involved in the cytokine signaling and/or phagosome function in neutrophils. gp49 is a member of the Ig superfamily and is expressed on mast cells, NK cells, and macrophages [43 44 45 46 47 ]. As the microarray analysis did not discriminate between two closely related mouse gp49 genes, gp49A and gp49B [44 ], we carried out RT-PCR analysis with primer sets specific for gp49A and gp49B, which revealed that the mRNA level of gp49B was about ten-fold higher than that of gp49A in the G-CSF-treated FDN1.1 cells (data not shown). The gp49B gene product has two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs) and is implicated in the negative regulation of IgE-activated mast cells [47 , 48 ].

Expression kinetics of the G-CSF-inducible genes in myeloid cell lines
To confirm the results of DNA microarray analysis and to characterize the time course of gene expression in detail, we carried out quantitative, real-time RT-PCR (Fig. 1 ) and Northern blot (Fig. 2 ) analyses. When the FDN1.1 cells were factor-starved and stimulated with IL-3, the expression of SOCS3 mRNA was induced weakly and rapidly disappeared (Figs. 1A and 2A) . In contrast, stimulation of FDN1.1 cells with G-CSF resulted in a strong expression of the SOCS3 gene. The SOCS3 mRNA level peaked 20 min after the stimulation and then declined, but a considerably high level of expression continued for more than 2 days. To compare the cytokine specificity of the SOCS family expression, we examined the expression of other SOCS members. As shown in Figure 2A , the mRNAs for SOCS1, SOCS2, and CIS1 were also induced by G-CSF, but their expression levels were much lower than those induced by IL-3, indicating that SOCS3 is preferentially up-regulated by G-CSF. Flotillin-1 and -2 were expressed at basal levels in FDN1.1 cells maintained in medium containing IL-3, and their mRNA levels were up-regulated three- to fivefold when cultured with G-CSF. The increase in flotillin mRNA levels was detected several hours after the G-CSF treatment, and high levels of expression were maintained thereafter (Figs. 1B and 1C and 2B) . Consistent with the increased expression of mRNA, the immunoblot analysis revealed that the levels of flotillin proteins gradually increased in FDN1.1 cells after G-CSF treatment (Fig. 2D) . The time-course of gp49B expression was similar to that of the flotillin genes. In contrast, CCR2 mRNA was not detectable at all when the FDN1.1 cells were cultured with IL-3. The onset of the CCR2 expression was detected at a relatively late time-point (12 h) after G-CSF stimulation, and its mRNA level kept increasing for at least 48 h. This expression profile is similar to that of the MPO gene (Figs. 1E and 1F and 2C) and suggests that the CCR2 gene is not a direct target of STAT3 but may rather be regulated by a differentiation-associated mechanism similar to the regulation of the MPO gene. A control analysis showed almost constant expression of the ß-actin gene, irrespective of culture conditions (Fig. 1G) .



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  		Figure 1. Kinetics of gene expression induced by G-CSF in FDN1.1 cells, which were stimulated with G-CSF ({blacksquare}) or IL-3 ({square}), and RNA was extracted at the indicated times (0, 20, and 40 min and 2, 4, 8, 12, 24, and 48 h). Expression levels of the indicated mRNAs were measured by real-time RT-PCR, as described in Materials and Methods.

 


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  		Figure 2. Detection of G-CSF-induced gene expression by Northern and Western blot analyses. (A–C) FDN1.1 cells were cultured in cytokine-free medium for 6 h and then stimulated with G-CSF or IL-3 for the indicated times. Total RNAs were analyzed by Northern blotting using probes for SOCS members (A), flotillin-1 and -2 (B), and CCR2 and MPO (C). CIS1, Cytokine-inducible SH2-containing protein 1. (D) FDN1.1 cells stimulated with G-CSF for the indicated times were lysed and analyzed by Western blotting using an anti-flotillin-1 or -2 antibody.

 
Previous studies have shown that various mIL-3-dependent myeloid precursor cell lines exhibit distinct phenotypes in response to G-CSF, probably depending on their respective differentiation stages [7 , 23 , 24 ]. To test whether the genes identified above were commonly regulated in other G-CSF-responsive hematopoietic cell lines, we analyzed their expression in the IL-3-dependent cell lines BA3-13, LG, and NFS60. The BA3-13 cell line, which was established by the forced expression of G-CSFR in the pro-B cell line Ba/F3, proliferates in response to G-CSF but does not express the MPO gene [23 , 24 ]. LG and NFS60 are murine myeloid cell lines expressing endogenous G-CSFR. When stimulated with G-CSF, LG cells differentiate into morphologically mature neutrophils, whereas NFS60 cells proliferate but do not differentiate [7 , 22 , 49 ]. As shown in Figure 3 , the SOCS3 mRNA was induced immediately (~20 min) upon G-CSF stimulation in all the cell lines tested here. The flotillin-1 gene showed basal expression when these cells were maintained with IL-3 and was only slightly up-regulated by G-CSF. The flotillin-2 and gp49B genes showed basal expression but no significant up-regulation by G-CSF (data not shown). In contrast, the CCR2 gene showed a consistent pattern in all the cell lines examined: a late onset of expression after G-CSF stimulation (~12 h) and a continual increase in the mRNA level. It is interesting that CCR2 mRNA was also induced by G-CSF in BA3-13 cells, in spite of the complete absence of MPO gene expression.



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  		Figure 3. Effect of G-CSF on gene expression in IL-3-dependent hematopoietic cell lines. BA3-13, LG, and NFS60 cells were stimulated with G-CSF, and expression of the indicated genes was analyzed as described in Figure 1 .

 
Gene expression profiles in myeloid progenitor cells during terminal differentiation
The above results suggest that the SOCS3 and CCR2 genes are generally up-regulated by G-CSF, but the expression of the flotillin and MPO genes is cell line-dependent. The MPO gene is expressed in promyelocytes but not in the cells at later stages of differentiation [50 , 51 ]. The fact that the G-CSF-induced activation of the MPO gene was not followed by a subsequent disappearance of the MPO mRNA in any cell line examined indicates that these cells stay at the promyelocytic or myelocytic stage and do not undergo terminal differentiation into mature neutrophils. To investigate the G-CSF-dependent gene expression during terminal neutrophilic differentiation, we used a recently developed immortalization system for myeloid progenitor cells. Calvo and co-workers [27 ] showed that the forced expression of HoxA9 in murine bone marrow cells immortalizes a late myelomonocytic progenitor in the presence of GM-CSF or IL-3 and that the immortalized progenitor cells can undergo terminal differentiation into mature neutrophils or macrophages when stimulated with G-CSF or M-CSF, respectively.

We established HoxA9-immortalized, GM-CSF-dependent myeloid progenitor cells according to their protocol and analyzed the G-CSF-dependent changes in gene expression by quantitative RT-PCR. As shown in Figure 4A , the immortalized bone marrow cells showed the typical morphology of immature progenitors when cultured in medium containing GM-CSF or IL-3 but exhibited neutrophilic differentiation within 72 h after the culture was shifted to a G-CSF-containing medium. In addition to the morphological change, expression of the neutrophil-specific surface marker Gr-1 was strongly induced by G-CSF, whereas the expression of the macrophage marker F4/80 was down-regulated (Fig. 4B) . These results indicate that the HoxA9-immortalized myeloid progenitors had the ability to terminally differentiate into mature neutrophils in response to G-CSF. Consistent with this observation, the expression of the MPO gene was high when the progenitor cells were cultured with GM-CSF or IL-3 and gradually decreased when the culture was shifted to the G-CSF-containing medium (Fig. 4C) . The expression of the SOCS3 gene was induced immediately by G-CSF, declined transiently, and again increased continuously. The flotillin-1, flotillin-2, and gp49B genes were expressed at basal levels when maintained with GM-CSF or IL-3 and were further activated by G-CSF stimulation. As observed in all the myeloid cell lines, expression of the CCR2 gene began much later than that of the other genes but continued to increase throughout the terminal differentiation. The clear contrast in the expression profiles of the CCR2 and MPO genes suggests that distinct mechanisms are involved in the regulation of their expression.



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  		Figure 4. Characterization of HoxA9-immortalized myeloid progenitor cells and the gene expression induced by G-CSF. (A) Myeloid progenitors immortalized by HoxA9 were cultured in medium containing GM-CSF, G-CSF, or IL-3 for 3 days and stained with Wright-Giemsa stain. (B) HoxA9-immortalized progenitor cells were cultured as in A, and the cell-surface expression of neutrophil marker Gr-1 (upper panels) and monocyte/macrophage marker F4/80 (lower panels) was examined by flow cytometry (filled histograms). Red lines indicate control staining with no antibody. Green lines in the middle and right panels indicate the staining of cells cultured in medium containing GM-CSF. (C) HoxA9-immortalized cells were cultured with GM-CSF ({square}), G-CSF ({blacksquare}), or IL-3 ({circ}) for the indicated times, and their gene expression was analyzed by real-time RT-PCR as described in Figure 1 .

 
Bone marrow neutrophils express functional CCR2
We finally examined whether normal neutrophils actually express functional CCR2. As shown in Figure 5A , RT-PCR analysis of mouse bone marrow cells revealed that Gr-1+ mature neutrophils and Gr-1– cells expressed significant levels of CCR2 mRNA (~1000 copies per ng total RNA). In vitro culture of the Gr-1– cells in the presence of G-CSF or GM-CSF induced Gr-1 expression in more than half of the cell population, suggesting terminal differentiation of a neutrophilic precursor in the Gr-1– population. It is interesting that G-CSF but not GM-CSF further up-regulated the CCR2 mRNA level in this process. Conversely, expression of CCR2 mRNA in Gr-1+ mature neutrophils did not increase further when cultured with G-CSF or GM-CSF.



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  		Figure 5. Bone marrow neutrophils express functional CCR2. (A) Mouse bone marrow cells were stained with PE-conjugated anti-Gr-1, and the PE-positive and -negative cells were sorted by FACSAria (BD Biosciences). The sorted cells were cultured in the presence of G-CSF (200 u/ml) or GM-CSF (100 u/ml) for 48 h. Total RNAs were extracted from cells before (time 0) and after (48 h) the in vitro culture and analyzed for CCR2 mRNA levels by real-time PCR (left panel). Expression of Gr-1 in cells before and after the in vitro culture was determined by flow cytometry (right panel). (B) Mouse bone marrow cells were assayed for the chemotactic activity toward the indicated concentration of MCP-1, as described in Materials and Methods. Each assay was done in triplicate, and data are representative of three independent experiments (left panel). In parallel, Gr-1 expression of the fresh bone marrow cells (before) and the migrated cells in the lower chambers (0, 0.3, and 1 nM MCP-1) was examined by flow cytometry (right panel).

 
We tested the chemotactic response of bone marrow neutrophils to MCP-1 by the Boyden chamber method. Since Gr-1 staining followed by flow cytometric fractionation of bone marrow cells resulted in impairment of their chemotactic activity, fresh bone marrow cells were used for the chemotaxis assay, and then, the migrated cells were characterized by Gr-1 staining. As shown in Figure 5B , MCP-1 induced significant migration of bone marrow cells compared with the control, random migration. The percentages of Gr-1+ cells in the migrated cells were similar (~70%) in all samples and were slightly higher than that in the bone marrow cells used for the assay (Fig. 5B , right panels), indicating that Gr-1+ cells as well as Gr-1– cells have an ability of migrating toward MCP-1. These results indicate that bone marrow neutrophils express functional CCR2.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this report, we investigated gene expression during the neutrophilic differentiation of myeloid progenitors in response to G-CSF. DNA microarray analysis of mRNAs from G-CSF-stimulated FDN-1.1 cells revealed several genes whose expression was strongly but differentially induced by G-CSF. Real-time, quantitative RT-PCR and Northern blot analyses revealed that the genes could be categorized into three groups based on the time-course of their induced expression: immediate-early, mid-early, and late.

The immediate-early genes, the SOCS3, JunB, and p19INK4D genes, are direct targets of STATs and do not require de novo protein synthesis for their expression [30 , 31 , 33 , 34 ]. Upon stimulation by G-CSF, the expression of SOCS3 is induced rapidly, reaches a peak within 20 min, and then declines swiftly, although it remains at a significant level thereafter. This is consistent with recent studies showing that SOCS3 is a negative-feedback regulator for G-CSF signaling and suppresses excess granulopoiesis [32 , 52 , 53 ]. Our results demonstrate that G-CSF preferentially induces SOCS3 expression, whereas IL-3 induces the expression of CIS1, SOCS1, and SOCS2, suggesting different modes of SOCS family gene expression control by cytokines.

The second group, which includes the genes for flotillin-1, flotillin-2, and the Ig-like receptor gp49B, shows a mid-early (2–4 h) onset of increased expression after G-CSF stimulation. These genes are expressed significantly at basal levels in all myeloid cells maintained in medium containing IL-3. Changes in their expression levels are marginal in some cell lines, but strong increases in their mRNA levels were observed in HoxA9-immortalized myeloid progenitors during G-CSF-induced neutrophil differentiation. A promoter analysis of the flotillin-1 gene showed that the 5'-flanking promoter region contains putative binding sequences for nuclear factors such as AML-1a and MZF-1/2A [54 ], suggesting that these myeloid-specific transcription factors may be involved in the G-CSF-regulated flotillin-1 expression.

The third group, represented by the CCR2 gene, showed a late onset (~12 h) of expression after G-CSF stimulation, and its mRNA level increased continuously in all the myeloid cells examined in this study. This time-course profile is quite different, not only from those of the former two groups but also from that of the MPO gene, whose expression is limited to the promyelocytic stage in vivo. The late onset of CCR2 gene expression suggests that this gene would be regulated by a second wave of G-CSF signaling where transcription factors other than STATs would play a principal role. Among the C/EBP family transcription factors that are known to play critical roles in myeloid differentiation [14 , 16 ], C/EBP-{alpha} has been suggested to play an important role in monocyte-specific expression of the CCR2 gene as well as neutrophil-specific expression of the MPO gene [16 , 55 ]. However, our results clearly indicate that distinct mechanisms are involved in the expression of these genes during G-CSF-induced neutrophilic differentiation. Our DNA microarray analysis showed that FDN1.1 cells express high levels of C/EBP{alpha}, C/EBPß, C/EBP{delta}, PU.1, NF-Y, and c-Myb, and their expression does not change significantly upon G-CSF stimulation, except for a threefold up-regulation of C/EBP{delta} (our unpublished observation). This observation suggests that the expression of these C/EBPs is not sufficient for activation of the MPO or CCR2 gene and that modification(s) such as phosphorylation may be involved in the regulation of C/EBP activity [16 , 56 ]. However, we cannot exclude the possibility that G-CSF may regulate expression of other myeloid-specific transcription factors such as C/EBP-{varepsilon}, which was not included in the gene entry of our microarray analysis.

Although we have not examined the physiological function of the newly identified G-CSF targets in neutrophils, some of them are likely to play important roles in the regulation of neutrophil functions. Flotillin-1 and -2 are enriched in the detergent-resistant membrane domains, lipid rafts [38 , 57 ], which have been implicated in various cellular processes, including receptor-mediated signal transduction, protein sorting, and vesicular trafficking [39 , 40 ]. Phagocytosis and phagosome formation constitutes one of the main membrane trafficking events in neutrophils. Recent studies have shown that flotillin-enriched lipid rafts are present on maturating phagosomes in the macrophage cell line J774 [39 ], monocytic cell line THP-1 [41 ], and neutrophils [57 ]. Thus, our results suggest that the up-regulation of flotillin proteins during G-CSF-induced differentiation may be required for the phagosome function in mature neutrophils.

The ITIM-containing, Ig-like receptor gp49B is expressed on mast cells and plays an inhibitory role in IgE- or cytokine-dependent mast cell activation and inflammation [43 , 48 , 58 ]. gp49B is also reported to be expressed inducibly in IL-2-stimulated NK cells and inhibits NK1.1-mediated cytokine production by NK cells [45 ]. A recent study using gp49B-deficient mice revealed that gp49B is expressed constitutively on neutrophils and is further up-regulated by lipopolysaccharide (LPS) administration and that this induced gp49B functions to prevent neutrophil-dependent vascular injury in response to LPS [59 ]. As the administration of LPS causes the abundant expression of G-CSF, it is likely in this case that the G-CSF induced by LPS in vivo up-regulates the expression of gp49B in neutrophils. Together with these studies, our results suggest that gp49B is expressed inducibly on neutrophils during G-CSF-mediated differentiation or activation and plays a role in the negative regulation of excess inflammatory response.

There has been little information about the expression and function of CCRs in neutrophils. Chemokines are classified into four groups, CXC, CC, C, and CX3C subfamilies, according to the number and spacing of conserved cysteine residues [35 ]. The CXC chemokine members have been characterized mainly as chemoattractants for neutrophils and T lymphocytes, whereas the CC chemokines are known to act on monocytes, eosinophils, basophils, NK cells, and T lymphocytes, but not on neutrophils [35 36 37 ]. Consistent with this, CCR2 null mice and MCP-1 null mice are reported to show a similar phenotype: the impaired recruitment of monocytes in several inflammatory model systems but normal behavior of neutrophils [60 , 61 ]. Our findings, however, indicate that bone marrow neutrophils are capable of expressing functional CCR2 during G-CSF-mediated differentiation, maturation, or activation and can be attracted by MCP-1. Previously, Johnston and co-workers [62 ] showed that MCP-1 could induce chemotaxis of neutrophils from adjuvant-immunized rats but not from naive rats, in vivo and in vitro, and that CCR1 and CCR2 were expressed only in neutrophils from the adjuvant-immunized rats. It is reasonable to speculate that the pre-existing inflammation triggered by the adjuvant injection might induce G-CSF, which in turn stimulates immature or mature neutrophils to express CCR2, which thus become responsive to MCP-1. Furthermore, a recent study showed that expression of CCR1, CCR2, and CCR5 was up-regulated in mouse blood neutrophils during sepsis induced by cecal ligation and puncture (CLP) [63 ]. As the CLP-induced polymicrobial sepsis causes strong expression of G-CSF in the lung and liver [64 ], the increased expression of these CCRs in blood neutrophils could be mediated by G-CSF. These studies, together with our results, suggest that G-CSF may modulate the chemokine responsiveness of neutrophils during these inflammatory responses. It was also shown that GM-CSF and interferon-{gamma} up-regulate CCR1 and CCR3 in human neutrophils, although the expression of other CCRs is not detectable [65 , 66 ]. We found that CCR1 was expressed inducibly in G-CSF-stimulated cells (our unpublished observation), suggesting that other CCRs may also be expressed inducibly in G-CSF- and/or GM-CSF-stimulated neutrophils. Analyses of the expression of CCRs and their function in neutrophils will be interesting issues to investigate in future studies.


   ACKNOWLEDGEMENTS
 
This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Akashi Izumi for excellent technical assistance. We are grateful to Dr. Toshio Kitamura (University of Tokyo) for the pMYs-IG retroviral vector and the packaging cell line Plat-E and to Dr. Akihiko Yoshimura (Kyushu University) for the mouse cDNAs for SOCS1, SOCS2, SOCS3, and CIS1.

Received September 15, 2004; revised March 7, 2005; accepted April 6, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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