Differential signaling mechanisms regulate expression of CC chemokine receptor-2 during monocyte maturation
© Phillips et al; licensee BioMed Central Ltd. 2005
Received: 15 December 2004
Accepted: 31 October 2005
Published: 31 October 2005
Peripheral blood monocytes and monocyte-derived macrophages are key regulatory components in many chronic inflammatory pathologies of the vasculature including the formation of atherosclerotic lesions. However, the molecular and biochemical events underlying monocyte maturation are not fully understood.
We have used freshly isolated human monocytes and the model human monocyte cell line, THP-1, to investigate changes in the expression of a panel of monocyte and macrophage markers during monocyte differentiation. We have examined these changes by RT-PCR and FACS analysis. Furthermore, we cloned the CCR2 promoter and analyzed specific changes in transcriptional activation of CCR2 during monocyte maturation.
The CC chemokine receptor 2 (CCR2) is rapidly downregulated as monocytes move down the macrophage differentiation pathway while other related chemokine receptors are not. Using a variety of biochemical and transcriptional analyses in the human THP-1 monocyte model system, we show that both monocytes and THP-1 cells express high levels of CCR2, whereas THP-1 derived macrophages fail to express detectable CCR2 mRNA or protein. We further demonstrate that multiple signaling pathways activated by IFN-γ and M-CSF, or by protein kinase C and cytoplasmic calcium can mediate the downregulation of CCR2 but not CCR1.
During monocyte-to-macrophage differentiation CCR2, but not CCR1, is downregulated and this regulation occurs at the level of transcription through upstream 5' regulatory elements.
Chemokines are a superfamily of small (8–10 kDa) proteins, which coordinate cellular responses to inflammation, insult or injury [1–4]. They also play a pivotal role in the regulation of leukocyte trafficking and extravasation through the luminal surface of endothelial cells into sites of tissue inflammation. The chemokine superfamily includes at least 20 receptors and more than 50 ligands [1–5]. The chemokine ligands can be separated into two major categories depending on whether they express a CC or CXC amino acid motif in their N-termini. This dichotomy appears to be functionally important since many CC chemokines preferentially target monocytes and T cells, while CXC chemokines such as IL-8 (CXCL8) tend to attract neutrophils. The CC chemokines bind to a family of G-protein coupled serpentine (seven transmembrane spanning) receptors, which are termed CC chemokine receptors (CCRs; [1, 3, 6]). Currently ten of the CC receptors have been identified and monocytes predominantly express three of them: CCR1, CCR2 and CCR5 [2, 7, 8]. These receptors can bind and signal to different CC chemokines including MCP-1 (CCL2), MIP-1α (CCL3) and RANTES (CCL3) [3, 4, 9] and these same chemokines are secreted by endothelial cells when activated by LDL or inflammatory cytokines [10–13] or when the endothelium is damaged [14, 15].
Indeed, the recruitment of peripheral blood monocytes to the site of injured endothelium by pro-inflammatory chemokines is a key regulatory component in the formation of an atherosclerotic lesion [16, 17]. The monocytes subsequently adhere to the endothelium and eventually migrate into the sub-intima [18, 19]. Here, they receive a series of differentiation signals including macrophage-colony stimulating factor (M-CSF) and minimally oxidized LDL that enables them to mature into macrophages. These macrophages then engulf large quantities of cholesterol to become lipid-laden foam cells. And it is the accumulation of these foam cells that eventually leads to the formation of characteristic fatty streaks, intermediate lesions and fibrous plaques [20, 21].
To date, though, the actual role of chemokines and their receptors in atherosclerosis has not been clearly established. However, recent studies using transgenic mouse models of atherosclerosis have provided convincing evidence that CCR2 is required for disease progression in apolipoprotein E-null mice [22, 23]. In these animals, disruption of the CCR2 gene greatly decreases lesion formation without affecting plasma lipid or lipoprotein concentrations. Using a slightly different approach Rollins and colleagues have demonstrated that CCL2, the ligand for CCR2, plays an equally important role in the development of atherosclerosis in low-density lipoprotein receptor deficient mice [24, 25]. Here, deletion of CCL2 leads to a significant reduction in lipid deposition within the aorta.
Despite the promising experimental results from these systems, relatively little is known about how the expression of chemokine receptor genes is regulated in normal or diseased human tissues. A recent paper by Yamamoto and colleagues  examined the basal regulatory mechanisms underlying expression of the CCR2 gene in the human monocyte cell line, THP-1. Indeed, this group characterized two key elements that seemed to be necessary and sufficient for the basal regulation of CCR2 expression: an Oct-1 binding sequence located 36 bp upstream of the TATA box and a tandem CAAT/enhancer-binding protein (C/EBP) binding sequence located, unusually, in the 5' UTR (at +50 to +77 bp). However, studies have not directly examined the molecular mechanisms by which basal expression of CCR2 is rapidly downregulated during the differentiation of monocytes into macrophages.
In an effort to address this issue, we have further developed a model of monocyte differentiation using THP-1 cells, which can be induced to mature into macrophages using either phorbol esters and ionomycin or a physiological combination of interferon-γ (IFN-γ) and M-CSF. In common with other studies, we report here that THP-1 cell maturation mediated by either high concentrations of PMA (50 nM) alone, or very low concentrations of PMA (1 nM) plus ionomycin (1 μM) is characterized by an increase in size, the development of an adherent phenotype and the up-regulation of a panel of differentiation markers [27–30]; in addition, CCR2, but not CCR1, was specifically down-regulated during differentiation. Modulation of CCR2 by PMA (50 nM), but not PMA (1 nM) plus ionomycin (1 μM), was found to be sensitive to inhibition by the broad-spectrum protein kinase inhibitor staurosporine. Furthermore, transient transfection of THP-1 cells with a CCR2-specific reporter construct indicated that PMA (50 nM) and PMA (1 nM) plus ionomycin (1 μM) mediated the downregulation of CCR2 through inhibition of CCR2-specific gene transcription. Moreover, physiological treatment of THP-1 monocytes with two known differentiation factors, IFN-γ and M-CSF, also promoted a differentiation phenotype essentially identical to that observed using pharmacologic stimuli. These data indicate that the activation of several intracellular signaling pathways selectively regulate the expression of CCR2 during monocyte maturation into macrophages.
Materials and methods
The THP-1 human monocytic cell line (ATCC) was grown in RPMI 1640 medium (GibcoBRL) containing 10 % fetal calf serum (FCS; GibcoBRL), 100 U/ml penicillin and 100 μg/ml streptomycin (GibcoBRL). The cells were maintained in culture at 37°C and 5% C02. Typically, cells (7 × 106 per point) were stimulated with 50 nM phorbol myristate acetate (PMA; Sigma) or 1 nM PMA plus 1 μM ionomycin (Calbiochem) in the presence or absence of the PKC inhibitor staurosporine (Calbiochem).
Isolation and culture of human peripheral blood monocytes
Peripheral blood mononuclear cells (PBMCs) were isolated from freshly prepared leukopacks (buffy coats) that were between 2–4 hours old. Briefly, 20 ml of blood from leukopacks were diluted using PBS (1:1) and layered over 15 ml of Ficoll-Paque PLUS (Amersham Pharmacia Biotech). Cells were then centrifuged at 400 × g for 20 minutes at room temperature. After this time, PBMCs were collected from the interphase and washed (× 2) with PBS and centrifuged at 150 × g for 10 minutes. Monocytes were further isolated from PBMCs using Percoll (Amersham Pharmacia Biotech) gradient centrifugation as previously described . Lipid staining of the monocytes revealed that their purity was greater than 90%. Finally, the cells were resuspended and cultured at 106/ml in RPMI 1640 supplemented with 10% autologous serum, penicillin and streptomycin (GibcoBRL).
Cloning the CCR2 promoter
A 1335 bp fragment of the promoter from the hCCR2 gene was cloned into the pGL3 vector (Promega) using sequences determined by Yamamoto and colleagues . This construct, termed pGL3-1335, contained the tandem C/EBP sites plus 1220 bp of the promoter sequence 5' of the transcriptional start site. The 5' primer contained a restriction site for kpnI, while the 3' primer contained a HindIII site. Each primer started with a 2 bp GC-rich clamp. The full primer sequences used are as follows:
pGL3-1335 5' CGGGTACCGCTGCTTTAGGTCCATTTACCCTC
pGL3-1335 3' GCAAGCTTATTGTACATTGGGTTGAGGTCTCC.
The genomic PCR was performed using an annealing temperature of 55°C (30 seconds) and an extension temperature of 72°C (2 minutes); 30 cycles of PCR were performed.
RNA isolation and RT-PCR
Total RNA was isolated using TRIzol (Life Technologies) and by following the manufacturer's instructions. Briefly, cells were lyzed in TRIzol and then mixed with chloroform. The lysate was then centrifuged to separate RNA, DNA and protein. Total RNA, which is contained in the upper aqueous phase was recovered and mixed with isopropanol to precipitate the RNA. The RNA was finally washed in 75% ethanol to remove impurities and dissolved in water.
5 μg of RNA prepared in this way was then taken and DNase treated to remove further enzymatic contamination, before being reverse transcribed to cDNA using a ProSTAR First Strand RT-PCR kit from Stratagene and by following the manufacturer's instructions.
Subsequently, RT-PCR was performed under standard conditions using primers specific for CCR1, CCR2 and GAPDH. The primer sequences used here were:
CCR1 sense 5'GAAACTCCAAACACCACAGAGGAC
CCR1 antisense 5'TTCGTGAGGAAAGTGAAGGCTG
CCR2 sense 5'CCACATCTCGTTCTCGGTTTATCAG
CCR2 antisense 5'CGTGGAAAATAAGGGCCACAG
CCR3 sense 5'CACTAGATACAGTTGAGACCTTTGG
CCR3 antisense 5'GGTAAAGAGCACTGCAAAGAGTC
CCR4 sense 5'ACCCCACGGATATAGCAGATACC
CCR4 antisense 5'CGTCGTGGAGTTGAGAGAGTACTTG
CCR5 sense 5'GGAGCCCTGCCAAAAAATC
CCR5 antisense 5'CTGTATGGAAAATGAGAGCTGC
CCR6 sense 5'TGGCAAGGGGTATAATTTGGG
CCR6 antisense 5'GACAGTCTGGTACTTGGGTTCACAG
CCR7 sense 5'AGACAGGGGTAGTGCGAGGC
CCR7 antisense 5'GGATGGAGAGCACTGTGGCTAG
CCR8 sense 5'ACCTCAGTGTGACAACAGTGACCG
CCR8 antisense 5'ACCATCTTCAGAGGCCACTTGG
CCR9 sense 5'CACTGAGGATGCCGATTCTGAG
CCR9 antisense 5'CGAAATCTGCGTGGCAGTTC
CXCR1 sense 5'CAGATCCACAGATGTGGGA
CXCR1 antisense 5'GTTTGGATGGTAAGCCTGG
CXCR2 sense 5'AACATGGAGAGTGACAGC
CXCR2 antisense 5'GATGAGTAGACGGTCCTTC
CXCR3 sense 5'TCCTTGAGGTGAGTGACCA
CXCR3 antisense 5'GTATTGGCAGTGGGTGGCG
CXCR4 sense 5'AGTATATACACTTCAGATAAC
CXCR4 antisense 5'CCACCTTTTCAGCCAACAG
CXCR5 sense 5'CTGGACAGATTGGACAACTA
CXCR5 antisense 5'CATCACAACAACTCCCTGA
GAPDH sense 5'TCCATGACAACTTTGGTATCG
GAPDH antisense 5'GTCGCTGTTGAAGTCAGAGGA
The annealing temperature used for RT-PCR was 55°C for 30 seconds and the extension temperature was 72°C for 1 minute; typically 30 cycles of PCR were performed. Under these conditions the product sizes for CCR1, CCR2 and GAPDH were 567 bp, 580 bp and 420 bp respectively.
Antibody staining and FACS analysis
THP-1 cells or PBMCs were resuspended in ice-cold staining buffer (PBS + 2% FCS + 0.1% sodium azide) and incubated with Fc block (Miltenyi Biotec) for 5 minutes at 4°C. Subsequently, primary antibodies were added (anti-CCR1, CCR2, CCR5, CCR7, CXCR2 and CXCR4; R&D Systems) at a final concentration of 0.5 μg/μl. The cells were then incubated at 4°C for 25 minutes, after which time they were washed twice in staining buffer. The secondary antibody used for these experiments was Alexa 488 (Molecular Probes) at a final concentration of 1 μg/μl. This time the cells were incubated at 4°C for 25 minutes in the dark. Following incubation with the secondary antibody, the cells were again washed twice, and then resuspended in 500 μl of staining buffer. Samples were finally analyzed on a FACScan flow cytometer (Becton Dickinson) using Cellquest 3.2.1f1 software. Peripheral blood monocytes, monocyte-derived macrophages and THP-1 cells were also stained for CD36, CD11b and CD68 (all purchased from BD Biosciences).
Transient transfection using DEAE/Dextran
THP-1 cells, grown to a density of 5–8 × 105/ml, were resuspended in Tris-buffered saline (TBS; 25 mM Tris.Cl, pH7.4, 137 mM NaCl, 5 mM KCl, 0.6 mM Na2 HPO4, 0.7 mM CaCl2 and 0.5 mM MgCl2). THP-1 cells (7 × 106 per point) were then added to 1 ml of TBS containing 5 μg of the CCR2 promoter-luciferase construct, 2 μg of the renilla control construct (pRL-SV40; Promega) and 500 μg/ml DEAE/Dextran (final concentration). This mixture was then left at room temperature for one hour. Next, DMSO was added to the cells drop-wise to a final concentration of 10% and incubated for 2 minutes at room temperature. Subsequently, the cells were washed twice in TBS, once in RPMI 1640 medium lacking FCS and antibiotics and once in RPMI 1640 complete medium. The cells were then resuspended in RPMI 1640 complete medium, stimulated with PMA and ionomycin (at the concentrations indicated) and finally incubated at 37°C and 5% CO2 for 48 hours.
After the 48-hour incubation period, cell extracts were made using the luciferase reporter lysis buffer (Promega). Each lysate was subsequently assayed in the dual luciferase reporter assay (Promega) following the manufacturer's instructions. Luciferase activity was determined using a Monolight series 2010 luminometer (Analytical Luminescence Laboratory) and then normalized to the renilla control.
Freshly isolated monocytes selectively downregulate CCR2, but not CCR1, in culture
These results indicate that one consequence of monocyte maturation is the selective downregulation of CCR2 gene expression followed by a loss of CCR2 protein from the surface of the cell. While the actual physiological role of this process is unknown, it is likely that CCR2 down-regulation may be involved in restricting 'reverse-migration' of differentiated monocytes back into the blood stream, and thus facilitating capture within the tissues.
PMA-treatment of monocytes induces selective downregulation of CCR2
Subsequently, we examined whether PMA modulated the cell surface expression of CCR1 and CCR2 by FACS analysis. THP-1 cells were again stimulated with PMA (50 nM) for the times indicated, before being stained with the appropriate antibodies and then analyzed by flow cytometry (Figure 2B). Whereas the levels of CCR1 remained high throughout the duration of the experiment, CCR2 protein expression decreased dramatically. The majority of the expression was lost by 24 hours and by 48 hours virtually no CCR2 was found on the surface of the cultured THP-1 cells (compare Figure 2B, left and right panels). Thus, THP-1 cells treated with PMA (50 nM) mimics the differentiation process observed in cultured monocytes.
Two distinct signal transduction pathways regulate CCR2 expression during monocyte maturation
Thus, these results identify at least two possible signal transduction pathways present in monocytes that could regulate the expression of CCR2 during monocyte differentiation.
CCR2 expression is regulated at the level of transcription
Having established that CCR2 is down-regulated during monocyte differentiation, we next wanted to determine whether the regulation occurs at the level of RNA stability or at the level of transcription. We, therefore, cloned a 1335 bp fragment of the CCR2 promoter using the sequence described by Yamamoto and colleagues . This fragment was then subcloned into the mammalian expression vector pGL3 upstream of the luciferase gene, generating the pGL3-1335 construct. In addition to the sequences upstream of the TATA box, pGL3-1335 included 115 bp of the 5'UTR, which contains the two tandem C/EBP repeats that are thought to be necessary for the basal expression of the CCR2 gene .
Treatment with IFN-γ and M-CSF produces a similar differentiation phenotype to that seen with PMA and ionomycin
The above results reflect a phenotype induced by pharmacologic agents and we next wanted to ensure that this phenotype is applicable to physiologic agents also. To that end, THP-1 cells treated with IFN-γ plus M-CSF have already been shown to promote monocyte maturation, although it has yet to be confirmed that these agents regulate CCR2 expression at the level of transcription . Initially, though, we wanted to demonstrate that monocytes treated with IFN-γ plus M-CSF showed changes in morphology similar to that observed with freshly isolated monocytes (compare Figures 1 and 6). After 48 hours treatment with IFN-γ plus M-CSF, monocytes became adherent and increased in size similar to that observed for freshly isolated monocytes in culture (compare Figure 1A and Figure 6A middle panel). PMA-treated monocytes also underwent similar changes in morphology (Figure 6A, lower panel). Furthermore, flow cytometric studies revealed that monocytes treated with either IFN-γ plus M-CSF or PMA strongly upregulated the macrophage maturation markers CD11b, CD36 and CD68 (Figure 6B). Similar results were observed for cells treated with PMA plus ionomycin (data not shown). Thus, monocytes treated with a panel of physiologic and pharmacologic stimuli promote maturation to the macrophage phenotype as determined by changes in morphology and upregulation of macrophage maturation markers.
Taken together, these data suggest that PMA (50 nM), PMA plus ionomycin and IFN-γ plus M-CSF mediate similar changes in the monocyte phenotype during maturation of these cells. Thus, the monocyte cell line, THP-1, is a useful model system with which to investigate the underlying regulatory mechanisms governing chemokine receptor expression during monocyte differentiation.
In this paper we demonstrate that a major consequence of monocyte maturation into macrophages is the selective downregulation of the chemokine receptor, CCR2, but not the related CCR1. We have further shown that there are multiple stimuli, which can selectively down-modulate CCR2 expression, including high concentrations of PMA (50 nM), or low PMA (1 nM) plus ionomycin (1 μM), or IFN-γ (500 U/ml) plus M-CSF (5 ng/ml). Each of these stimuli regulate the expression of CCR2 at the level of transcription, although it appears that at least two different signal transduction pathways are involved based on the ability of staurosporine to interfere with these processes. Treatment of THP-1 monocytes with staurosporine abrogated the ability of PMA and IFN-γ plus M-CSF to downregulate CCR2. By contrast, staurosporine was unable to block PMA plus ionomycin mediated downregulation of CCR2 expression. Thus, this study provides evidence that there is dynamic and selective regulation of the CCR2 gene during monocyte differentiation.
Our results indicate that treatment of THP-1 cells with either PMA alone (50 nM) or PMA (1 nM) plus ionomycin (1 μM) promotes a differentiation phenotype that is characterized by morphological changes and altered CCR2 gene expression. Indeed, these observations have already been noted by other researchers studying monocyte differentiation [27, 28, 32]. In particular, we show that THP-1 cells rapidly become adherent and their morphology changes from the typical round shape of monocytes to spindle-shaped cells with pseudopodia, which are characteristic of macrophages. At the same time there was also an increase in the size and granularity of the cells. In addition, we demonstrated an up-regulation in expression of genes associated with monocyte differentiation, notably CD11b, CD36 and CD68. Concomitantly, the expression of CCR2, but not CCR1, was selectively downregulated, suggesting that the loss of this chemokine receptor is a consequence of monocyte differentiation. This downregulation was observed at the level of cell surface receptor expression, mRNA expression, and transcription. Clearly, these are specific regulatory events since the levels of CCR1 mRNA are not affected by either combination of pharmacologic agents.
However, when THP-1 cells were treated with PMA (50 nM) or PMA plus ionomycin in the presence of staurosporine, differential results were obtained: PMA-mediated modulation of CCR2 was sensitive to the inhibitory effects of staurosporine (50 nM), whereas staurosporine concentrations as high as 200 nM failed to block PMA plus ionomycin-induced downregulation of CCR2. Staurosporine alone did not promote the loss of either CCR2 or CCR1. These results indicate that staurosporine defines a dichotomy in the regulation of CCR2 expression by PMA (50 nM) versus PMA plus ionomycin that had not previously been appreciated.
Staurosporine, itself, is a broad-spectrum inhibitor of protein kinases including PKA, PKC, and PKG. PMA has classically been shown to act almost exclusively through PKC and this would explain why staurosporine was able to block the PMA-induced downregulation of CCR2. By inference, PMA plus ionomycin would appear to act through a signal transduction pathway that is not inhibited by staurosporine and presumably this means that second messengers other than PKA, PKC and PKG are involved. To that end, calcineurin, a calcium-sensitive phosphatase may be a target for PMA plus ionomycin . An increase in the intracellular calcium concentration (such as that afforded by the presence of ionomycin) promotes a conformational change in calcineurin, which then dephosphorylates and activates the transcription factor NFAT facilitating its translocation to the nucleus. In addition, it has been shown that PMA enhances the calcium sensitivity of NFAT, thus creating a synergistic signal [33, 34]. This synergy may result from de novo synthesis and post-translational modification of another transcription factor termed activating protein-1, AP-1 [33, 34]. Indeed, NFAT proteins show a characteristic ability to co-operate with AP-1 in DNA-binding and transactivation [33, 34]. Interestingly, in the region of the CCR2 promoter that we cloned there are two putative binding sites for AP-1 (core binding motif TGA(C/G)TCA) and three putative binding sites for NFAT (core binding motif GGAAA) as determined by the MatInspecter transcription factor binding site analysis program. It has also been suggested that additional transcription factors including OCT1 and C/EBP can act synergistically with NFAT and again there are multiple binding sites for each of these DNA-binding proteins in the CCR2 promoter, although at this stage we have no evidence to suggest that they are involved in the physiological regulation of CCR2 gene expression.
A requirement for co-operation and cross-talk between these two pharmacologic agents is further supported by the fact that ionomycin alone (at concentrations as high as 1 μM) was unable to down-modulate CCR2.
Some reports have suggested that CCL2 could be involved in the early stages of CCR2 protein down-modulation, while other studies indicate that the differentiation process itself, is a major factor in the selective loss of CCR2 gene expression [8, 32]. Numerous cytokines are known to be involved in monocyte activation and differentiation, among them M-CSF and IFN-γ [32, 35, 36]. M-CSF is a lineage-specific hematopoetic growth factor that stimulates monocyte differentiation [35, 36]. The c-fms proto-oncogene encodes a high affinity receptor for M-CSF  and it has been shown that THP-1 cells express this protein and that it is up-regulated during differentiation. However, cells stimulated with M-CSF alone for 48 hours did not lose expression of CCR2 (data not shown).
Conversely, IFN-γ alone, which is constitutively expressed by monocyte lineage cells and which promotes maturation of monocytes to macrophages , did significantly reduce expression of CCR2, although the cells did not become adherent and neither did they change their morphology (data not shown). Interestingly, IFN-γ has been demonstrated to up-regulate levels of M-CSF in monocytes during maturation  and when both IFN-γ and M-CSF were added, THP-1 cells did become adherent, changed their morphology and selectively lost CCR2, but not CCR1 – all of which are characteristics of the monocyte differentiation phenotype. These results are in keeping with the studies published by Tangirala and colleagues, who reported similar phenomena in THP-1 cells . In addition, our studies also demonstrated that the regulatory effects mediated by IFN-γ plus M-CSF occurred at the level of transcription, where a significant down-regulation in CCR2 promoter activity was observed. Moreover, in the presence of staurosporine, IFN-γ plus M-CSF was unable to down-regulate levels of CCR2. This result probably reflects the fact that IFN-γ signals extensively through the JAK-STAT pathway, and studies have suggested that staurosporine can block phosphorylation of Janus kinases [39, 40]. In addition, we have found two putative binding sites in the CCR2 promoter for STAT transcription factors which would further support the contention that these transcription factors may be important in the regulation of IFN-γ mediated downregulation of CCR2.
This study demonstrates that expression of the chemokine receptor CCR2 is exquisitely correlated with monocyte maturation. Freshly isolated monocytes express high levels of both CCR2 RNA and protein, whereas monocyte-derived macrophages express neither CCR2 RNA nor protein. Conversely, levels of the closely-related chemokine receptor CCR1 remained stable and elevated throughout monocyte maturation. An analysis of the biochemical and molecular mechanisms underlying the regulated expression of CCR2 revealed the existence of several signaling pathways that selectively down-modulate CCR2 gene expression during monocyte differentiation; this expression was largely regulated at the level of transcription. Signaling through PMA and IFN-γ plus M-CSF, but not PMA plus ionomycin was abrogated by prior treatment of the THP-1 cells with staurosporine. Although the physiological role of this process is not well understood, it is likely that CCR2 down-regulation may be involved in restricting the 'reverse-migration' of differentiated monocytes back into the blood stream. This in turn facilitates the retention of differentiated monocytes within inflamed tissues. Thus, by improving our understanding of the regulatory mechanisms that govern CCR2 expression on monocyte lineage cells, we can better appreciate how monocyte recruitment and activation is controlled during chronic inflammatory pathologies such as atherosclerosis.
- Rossi D, Zlotnik A: The biology of chemokines and their receptors. Annu Rev Immunol. 2000, 18: 217-242. 10.1146/annurev.immunol.18.1.217.View ArticlePubMedGoogle Scholar
- Murphy PM, Baggiolini M, Charo IF, Hebert CA, Horuk R, Matsushima K, Miller LH, Oppenheim JJ, Power CA: International union of pharmacology. XXII. Nomenclature for chemokine receptors. Pharmacol Rev. 2000, 52: 145-176.PubMedGoogle Scholar
- Premack BA, Schall TJ: Chemokine receptors: gateways to inflammation and infection. Nat Med. 1996, 2: 1174-1178. 10.1038/nm1196-1174.View ArticlePubMedGoogle Scholar
- Baggiolini M: Chemokines and leukocyte traffic. Nature. 1998, 392: 565-568. 10.1038/33340.View ArticlePubMedGoogle Scholar
- Murphy PM: International Union of Pharmacology. XXX. Update on chemokine receptor nomenclature. Pharmacol Rev. 2002, 54: 227-229. 10.1124/pr.54.2.227.View ArticlePubMedGoogle Scholar
- Power CA, Wells TN: Cloning and characterization of human chemokine receptors. Trends Pharmacol Sci. 1996, 17: 209-213. 10.1016/0165-6147(96)10019-5.View ArticlePubMedGoogle Scholar
- Charo IF, Myers SJ, Herman A, Franci C, Connolly AJ, Coughlin SR: Molecular cloning and functional expression of two monocyte chemoattractant protein 1 receptors reveals alternative splicing of the carboxyl-terminal tails. Proc Natl Acad Sci U S A. 1994, 91: 2752-2756.PubMed CentralView ArticlePubMedGoogle Scholar
- Fantuzzi L, Borghi P, Ciolli V, Pavlakis G, Belardelli F, Gessani S: Loss of CCR2 expression and functional response to monocyte chemotactic protein (MCP-1) during the differentiation of human monocytes: role of secreted MCP-1 in the regulation of the chemotactic response. Blood. 1999, 94: 875-883.PubMedGoogle Scholar
- Neote K, DiGregorio D, Mak JY, Horuk R, Schall TJ: Molecular cloning, functional expression, and signaling characteristics of a C-C chemokine receptor. Cell. 1993, 72: 415-425. 10.1016/0092-8674(93)90118-A.View ArticlePubMedGoogle Scholar
- Brown Z, Gerritsen ME, Carley WW, Strieter RM, Kunkel SL, Westwick J: Chemokine gene expression and secretion by cytokine-activated human microvascular endothelial cells. Differential regulation of monocyte chemoattractant protein-1 and interleukin-8 in response to interferon-gamma. Am J Pathol. 1994, 145: 913-921.PubMed CentralPubMedGoogle Scholar
- Goebeler M, Yoshimura T, Toksoy A, Ritter U, Brocker EB, Gillitzer R: The chemokine repertoire of human dermal microvascular endothelial cells and its regulation by inflammatory cytokines. J Invest Dermatol. 1997, 108: 445-451. 10.1111/1523-1747.ep12289711.View ArticlePubMedGoogle Scholar
- Marfaing-Koka A, Devergne O, Gorgone G, Portier A, Schall TJ, Galanaud P, Emilie D: Regulation of the production of the RANTES chemokine by endothelial cells. Synergistic induction by IFN-gamma plus TNF-alpha and inhibition by IL-4 and IL-13. J Immunol. 1995, 154: 1870-1878.PubMedGoogle Scholar
- Martin T, Cardarelli PM, Parry GC, Felts KA, Cobb RR: Cytokine induction of monocyte chemoattractant protein-1 gene expression in human endothelial cells depends on the cooperative action of NF-kappa B and AP-1. Eur J Immunol. 1997, 27: 1091-1097.View ArticlePubMedGoogle Scholar
- Kumar AG, Ballantyne CM, Michael LH, Kukielka GL, Youker KA, Lindsey ML, Hawkins HK, Birdsall HH, MacKay CR, LaRosa GJ, Rossen RD, Smith CW, Entman ML: Induction of monocyte chemoattractant protein-1 in the small veins of the ischemic and reperfused canine myocardium. Circulation. 1997, 95: 693-700.View ArticlePubMedGoogle Scholar
- Wysocki SJ, Zheng MH, Smith A, Lamawansa MD, Iacopetta BJ, Robertson TA, Papadimitriou JM, House AK, Norman PE: Monocyte chemoattractant protein-1 gene expression in injured pig artery coincides with early appearance of infiltrating monocyte/macrophages. J Cell Biochem. 1996, 62: 303-313. 10.1002/(SICI)1097-4644(199609)62:3<303::AID-JCB1>3.0.CO;2-V.View ArticlePubMedGoogle Scholar
- Ross R: The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature. 1993, 362: 801-809. 10.1038/362801a0.View ArticlePubMedGoogle Scholar
- Berliner JA, Navab M, Fogelman AM, Frank JS, Demer LL, Edwards PA, Watson AD, Lusis AJ: Atherosclerosis: basic mechanisms. Oxidation, inflammation, and genetics. Circulation. 1995, 91: 2488-2496.View ArticlePubMedGoogle Scholar
- Cai JP, Hudson S, Ye MW, Chin YH: The intracellular signaling pathways involved in MCP-1-stimulated T cell migration across microvascular endothelium. Cell Immunol. 1996, 167: 269-275. 10.1006/cimm.1996.0035.View ArticlePubMedGoogle Scholar
- Randolph GJ, Furie MB: A soluble gradient of endogenous monocyte chemoattractant protein-1 promotes the transendothelial migration of monocytes in vitro. J Immunol. 1995, 155: 3610-3618.PubMedGoogle Scholar
- Ross R: Cell biology of atherosclerosis. Annu Rev Physiol. 1995, 57: 791-804. 10.1146/annurev.ph.57.030195.004043.View ArticlePubMedGoogle Scholar
- Lusis AJ: Atherosclerosis. Nature. 2000, 407: 233-241. 10.1038/35025203.PubMed CentralView ArticlePubMedGoogle Scholar
- Boring L, Gosling J, Cleary M, Charo IF: Decreased lesion formation in CCR2-/- mice reveals a role for chemokines in the initiation of atherosclerosis. Nature. 1998, 394: 894-897. 10.1038/29788.View ArticlePubMedGoogle Scholar
- Charo IF, Peters W: Chemokine receptor 2 (CCR2) in atherosclerosis, infectious diseases, and regulation of T-cell polarization. Microcirculation. 2003, 10: 259-264. 10.1038/sj.mn.7800191.View ArticlePubMedGoogle Scholar
- Gu L, Okada Y, Clinton SK, Gerard C, Sukhova GK, Libby P, Rollins BJ: Absence of monocyte chemoattractant protein-1 reduces atherosclerosis in low density lipoprotein receptor-deficient mice. Mol Cell. 1998, 2: 275-281. 10.1016/S1097-2765(00)80139-2.View ArticlePubMedGoogle Scholar
- Rollins BJ: Chemokines and atherosclerosis: what Adam Smith has to say about vascular disease. J Clin Invest. 2001, 108: 1269-1271. 10.1172/JCI200114273.PubMed CentralView ArticlePubMedGoogle Scholar
- Yamamoto K, Takeshima H, Hamada K, Nakao M, Kino T, Nishi T, Kochi M, Kuratsu J, Yoshimura T, Ushio Y: Cloning and functional characterization of the 5'-flanking region of the human monocyte chemoattractant protein-1 receptor (CCR2) gene. Essential role of 5'-untranslated region in tissue-specific expression. J Biol Chem. 1999, 274: 4646-4654. 10.1074/jbc.274.8.4646.View ArticlePubMedGoogle Scholar
- Tontonoz P, Nagy L, Alvarez JG, Thomazy VA, Evans RM: PPARgamma promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell. 1998, 93: 241-252. 10.1016/S0092-8674(00)81575-5.View ArticlePubMedGoogle Scholar
- Rovera G, Santoli D, Damsky C: Human promyelocytic leukemia cells in culture differentiate into macrophage-like cells when treated with a phorbol diester. Proc Natl Acad Sci U S A. 1979, 76: 2779-2783.PubMed CentralView ArticlePubMedGoogle Scholar
- Naito M, Umeda S, Yamamoto T, Moriyama H, Umezu H, Hasegawa G, Usuda H, Shultz LD, Takahashi K: Development, differentiation, and phenotypic heterogeneity of murine tissue macrophages. J Leukoc Biol. 1996, 59: 133-138.PubMedGoogle Scholar
- Yesner LM, Huh HY, Pearce SF, Silverstein RL: Regulation of monocyte CD36 and thrombospondin-1 expression by soluble mediators. Arterioscler Thromb Vasc Biol. 1996, 16: 1019-1025.View ArticlePubMedGoogle Scholar
- Seager Danciger J, Lutz M, Hama S, Cruz D, Castrillo A, Lazaro J, Phillips R, Premack B, Berliner J: Method for large scale isolation, culture and cryopreservation of human monocytes suitable for chemotaxis, cellular adhesion assays, macrophage and dendritic cell differentiation. J Immunol Methods. 2004, 288: 123-134. 10.1016/j.jim.2004.03.003.View ArticlePubMedGoogle Scholar
- Tangirala RK, Murao K, Quehenberger O: Regulation of expression of the human monocyte chemotactic protein-1 receptor (hCCR2) by cytokines. J Biol Chem. 1997, 272: 8050-8056. 10.1074/jbc.272.12.8050.View ArticlePubMedGoogle Scholar
- Rao A, Luo C, Hogan PG: Transcription factors of the NFAT family: regulation and function. Annu Rev Immunol. 1997, 15: 707-747. 10.1146/annurev.immunol.15.1.707.View ArticlePubMedGoogle Scholar
- Macian F, Lopez-Rodriguez C, Rao A: Partners in transcription: NFAT and AP-1. Oncogene. 2001, 20: 2476-2489. 10.1038/sj.onc.1204386.View ArticlePubMedGoogle Scholar
- Lenny N, Westendorf JJ, Hiebert SW: Transcriptional regulation during myelopoiesis. Mol Biol Rep. 1997, 24: 157-168. 10.1023/A:1006859700409.View ArticlePubMedGoogle Scholar
- Clarke S, Gordon S: Myeloid-specific gene expression. J Leukoc Biol. 1998, 63: 153-168.PubMedGoogle Scholar
- Nienhuis AW, Bunn HF, Turner PH, Gopal TV, Nash WG, O'Brien SJ, Sherr CJ: Expression of the human c-fms proto-oncogene in hematopoietic cells and its deletion in the 5q- syndrome. Cell. 1985, 42: 421-428. 10.1016/0092-8674(85)90099-6.View ArticlePubMedGoogle Scholar
- Scheibenbogen C, Andreesen R: Developmental regulation of the cytokine repertoire in human macrophages: IL-1, IL-6, TNF-alpha, and M-CSF. J Leukoc Biol. 1991, 50: 35-42.PubMedGoogle Scholar
- Fiorucci G, Percario ZA, Marcolin C, Coccia EM, Affabris E, Romeo G: Inhibition of protein phosphorylation modulates expression of the Jak family protein tyrosine kinases. J Virol. 1995, 69: 5833-5837.PubMed CentralPubMedGoogle Scholar
- Callus BA, Mathey-Prevot B: Interleukin-3-induced activation of the JAK/STAT pathway is prolonged by proteasome inhibitors. Blood. 1998, 91: 3182-3192.PubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.