Chemokines are a family of small cytokines or proteins secreted by cells that act as a chemoattractant to guide cell migration (1, 2). Chemokine receptors are G-protein-coupled, seven-transmembrane receptors (2). Among these receptors, the monocyte chemoattractant protein-1 (MCP-1)/CCR2 system is an important chemokine and chemokine receptor in the migration of monocytes and macrophages (3, 4). MCP-1 is known to stimulate leukocyte chemotaxis to inflammatory sites, such as those in rheumatoid arthritis, atherosclerosis, asthma, and chronic rejection of renal transplantation (5–7), and is present and elevated in these diseases. Treatment with MCP-1 neutralizing antibodies or other biologic antagonists has reduced inflammation in a number of animal models (8, 9). Therefore, this suggests that modulation of MCP-1 expression will be beneficial in the treatment of inflammatory diseases. CCR2 antagonist is a receptor antagonist for MCP-1, blocks the MCP-1/CCR2 system, and is known to be a potential anti-inflammatory therapeutic agent. It has recently been reported that CCR2 antagonist can reduce the symptoms and histopathology in inflammatory diseases such as arthritis (10).
Recently optimized labeling techniques for superparamagnetic iron oxide have improved the efficiency of macrophage labeling and have allowed the ex vivo labeling of cells (11). Using this technique, macrophage homing and the rate of macrophage recruitment toward the infection site can be observed with in vivo MRI (12–14). The purposes of this study are to assess the effect of CCR2 antagonist on microphage migration and to evaluate the feasibility of in vivo MRI to assess the inhibition of chemotactic activity by the CCR2 antagonist.
MATERIALS AND METHODS
CCR2 Antagonist and Cell Lines
For the CCR2 antagonist, RS-102895 hydrochloride (C21H21F3N2O2.HCl; Sigma, St. Louis, MO) was used. RS-102895 is a chemokine receptor CCR2 antagonist which belongs to the spiropiperidine class. It is potent and specific for the CCR2b receptor (3). The emulsion of 40-mM RS-102895 hydrochloride was made from 5 mg RS-102895 hydrochloride, which was reconstituted with 292.8 mL dimethyl sulfoxide (DMSO). For the control group, phosphate-buffered saline (PBS) containing 2.5% ethanol and 2.5% DMSO without CCR2 antagonist was used.
For the mouse macrophages, RAW 264.7 cells were obtained from the American Type Culture Collection (No. TIB-71™) and were maintained in Dulbecco's modified Eagle's medium (GIBCO, Carlsbad, CA) with 1500-mM sodium bicarbonate and high glucose, respectively, and containing 10% fetal bovine serum (GIBCO) and 1% penicillin-streptomycin-neomycin (GIBCO). The cells were cultured in standard atmosphere.
The binding ability was assessed by the method that has been previously described (15, 16). Briefly, RAW 264.7 cells were washed with PBS (GIBCO) before being resuspended in binding buffer (Dulbecco's modified Eagle's medium with 0.5% bovine serum albumin; Sigma, St. Louis, MO) and 25-mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH = 7.4, Sigma) at 1 × 105 cells/200 μL. For saturation binding analysis, cells were incubated with murine recombinant MCP-1 (mMCP-1; R&D Systems, Inc., Minneapolis, MN) in indicated concentrations from 0.1 to 5 ng/mL for 30 min at 4°C, were then washed twice, and were incubated with rat antimouse MCP-1 immunoglobulin G2B (IgG2B) (clone # 123602; R&D Systems, Inc.) for 30 min on ice. The cells were then washed with PBS and were incubated with fluorescein isothiocyanate–conjugated goat antirat IgG, F (ab′)2 (#112-096-072; Jackson ImmunoResearch Laboratories, Ltd., Suffolk, UK) for 30 min on ice. The cells were then washed and resuspended in PBS for analysis using fluorometer (Spectra Max Gemini XS; Molecular Devices Inc., Sunnyvale, CA). To determine whether 20-mM doses of CCR2 antagonist inhibit the binding affinity, expressed CCR2 receptor on RAW 264.7 cells was measured using the MCP-1 saturation binding assay. mMCP-1 bound to RAW 264.7 cells was evaluated in vitro by assessing the fluorescence density, and equilibrium binding constant was then calculated.
Expression of mRNA of CCR2 and CD11b
To analyze messenger ribonucleic acid (mRNA) expression levels by reverse transcription–polymerase chain reaction (PCR), total RNA was extracted from RAW 264.7 cells using Trizol reagent (Invitrogen Inc., Carlsbad, CA). 1 μg of total RNA from each sample was reverse transcribed. Expression mRNA for CCR2 and CD11b by mMCP-1 in RAW264.7 was estimated using conventional PCR with and without the CCR2 antagonist. The following specific primers were used in reverse transcription–PCR: mouse CCR2, sense 5′-AAG AGG GCA TTG GAT TCA CC-3′, antisense 5′-TCC CTC CTT CCC TGC TTA AA-3′; mouse CD11b, sense 5′-ACG TGC TCA AGT TAA CCG AC-3′, antisense 5′-ACT CGA TGC TCC ATT ACT GC-3′; mouse MCP-1, sense 5′-TGC TGA CCC CAA GAA GGA AT-3′, antisense 5′-CCT TAG GGC AGA TGC AGT TT-3′. As an internal standard, glyceraldehyde 3-phosphate dehydrogenase was amplified and analyzed under identical conditions, using the following specific primers: sense 5′-GAC CCC TTC ATT GAC CTC-3′ and antisense 5′-GCT AAG CAG TTG GTG GTG-3′. PCRs involved 25 to 33 cycles, and each cycle was at 94°C for 30 sec, 60°C for 1 min, and 72°C for 1 min. PCR products were electrophoresed on ethidium bromide (Sigma)–containing agarose gels, and the band intensity was measured by densitometry scanning using the Versa Doc Imaging System (Bio Rad Inc., Hercules, CA).
Expression of CCR2 and CD11b on the Cell Surface
Fluorescence-activated cell sorting was used to detect CD11b and CCR2 protein on the cell surface. RAW 264.7 cells were labeled with rabbit antimouse CKR-2 polyclonal IgG (1:50) (M-50; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) in order to detect CCR2 or with rat antimouse Integrin αM/CD11b IgG2B (0.25 μg; 1 × 105 cells) (clone: M1/70, R&D Systems, Inc.) in order to detect CD11b. The CKR-2 polyclonal IgG were detected using Cy3-conjugated goat antirabbit IgG, F(ab′)2 (1:200) (#111-166-006; Jackson Immunoresearch Europe Ltd.). The CD11b antibodies were detected using Cy3-conjugated goat anti-rat IgG, F(ab′)2 (1:200) (#112-166-072; Jackson Immunoresearch Europe Ltd.). Data were analyzed according to the mean fluorescence intensity (MFI) of cell labeled antibodies by FACScan (BD Biosciences, San Jose, CA).
Confocal Microscopy for Expression of CCR2 and CD11b
After RAW 264.7 cells were preincubated with/without 10 μM CCR2 antagonist for 30 min, the cells were incubated with 5 or 10 ng/mL mMCP-1 for 24 h. Those cells were plated on slide glass using cytospin (AutoSmear; Sakura Finetek Inc., Tokyo, Japan), fixed with 3.7% paraformaldehyde in PBS for 10 min at 37°C and were then blocked with PBS containing 0.8 μg/mL IgG Fc portion (Jackson Immunoresearch Ltd.) and 0.1% bovine serum albumin (Sigma). RAW 264.7 cells were labeled with antimouse Integrin αM/CD11b IgG2B, and the mouse CD11b antibodies were detected with Cy3-conjugated goat antirat IgG, F(ab′)2 (1:200). Coverslips were mounted on glass microscope slides with Dako fluorescent mounting medium (Dako Corp., Carpinteria, CA). Images were captured by confocal microscopy using a TCS-SP2 system (Leica Microsystems, Wetzlar, Germany).
Experimental Groups for Migration Test
Labeled macrophages were divided into three groups (negative control, CCR2 antagonist, and positive control). The negative control group was incubated with PBS (GIBCO) for 30 min. The experimental CCR2 antagonist group was incubated with 1 μM CCR2 antagonist for 30 min. The positive control group was incubated with pertussis toxin (P 7208, Sigma), which blocks all G-protein-coupled receptors.
In Vitro Migration Test
To access the effect of the CCR2 antagonist for cell migration in vitro, a migration assay using a transwell plate (Transwell® #3428; Corning Inc., Corning, NY) was performed. Macrophages (5 × 105) were stained with fluorescence probes, 1,1 didodecyl-3,3,3',3-'tetramethylindocarbocyanine perchlorate (Molecular Probe® V22885; Invitrogen Inc.) or 3,3′-dioctadecyloxacarbocyanine perchlorate (Molecular Probe® V22886; Invitrogen Inc.) and were pretreated with PBS, CCR2 antagonist, or pertussis toxin, according to the groups.
The macrophages treated with CCR2 antagonist and those treated with PBS were applied in the upper chamber of the transwell plate. In the lower chamber, 200 ng/mL mMCP-1 mixed with growth factor reduced Matrigel™ (BD biosciences, San Jose, CA) was added and incubated at 37°C for 24 h. After 24 h, the macrophages that had migrated toward the lower chamber were captured by confocal microscopy, using a TCS-SP2 system (Leica Microsystems).
Animals and Experimental Abscess Model
Our study was performed according to the guidelines of the National Institutes of Health and the recommendations of the institution's committee on animal research. The protocol was approved by the local institutional review committee on animal care.
The study included 12 male C3H/HeN (OrientBio, Sungnam, Korea) mice (weight range, 20-25 g). All animals were kept in cages with standardized lighting conditions and free access to water and food. While the mice were receiving general inhalation anesthetic (1.5% isoflurane in a 1:2 mixture of O2/N2O; Abbott Laboratories, Abbott Park, IL), a unilateral, left-sided, deep-calf-muscle abscess was induced by means of intramuscular inoculation of 0.05 mL of a bacterial suspension (Staphylococcus aureus, # 25923; American Type Culture Collection, Manassas, VA). The inoculations were performed with a 30-gauge needle. Bacterial suspensions were prepared by washing the organisms once with 0.9% saline, resuspending them in PBS without calcium and magnesium (PBS; GIBCO Laboratories, Grand Island, NY), and diluting them to 2 × 109 colony-forming units per milliliter. All animals developed an abscess in the left calf muscle within 48 h of bacterial inoculation. No clinical signs of systemic infection were observed.
For MRI, macrophages were incubated with superparamagnetic iron oxide (Ferridex IV; Advanced Magnetics, Cambridge, MA) in a concentration of 112 μgFe/mL for 24 h in standard tissue culture incubators, according to the previously described methods (13). Macrophages with phagocytosed superparamagnetic iron oxide adhered to the tissue culture flasks, which allowed free superparamagnetic iron oxide to be easily removed by gentle washing with PBS. The purified, adhered, and labeled macrophages were then lifted off the plate by incubating them with 10 mmol/L ethylenediaminetetraacetic acid in PBS without calcium and magnesium for 10 min at 37°C. Macrophages were then washed with PBS and were resuspended in Hanks' balanced salt solution (Gibco Laboratories), with a dilution of 2 × 106 cells per milliliter, before intravenous injection.
In Vivo MR Evaluation of the Migration Test and Histologic Examination
Treated and labeled macrophages (6 × 105) were injected through the tail vein of 12 mice in three groups 48 h after bacterial inoculation. Each group consisted of four mice. The infected left calf was imaged using a 4.7-T Bruker Biospin imager (Bruker Medical Systems, Karlsruhe, Germany). All animals were anesthetized (1.5% isoflurane in a 1:2 mixture of O2/N2O) and were imaged 1 day following intravenous injection of macrophages. The imaging protocol included a T*2-weighted, gradient-echo sequence (pulse repetition time/echo time, 356.5/10.3 msec; flip angle, 30°). The spatial resolution was 256 × 256 matrix; field of view, 2.18 × 2.06 cm; section thickness, 0.67mm; section gap, 0.33mm; and number of sections, 16.
Signal intensities (SI) were measured within regions of interest (ROIs) on T*2-weighted images. The six ROIs (12 pixel units) were placed within the cellular infiltrate of the abscess wall (three ROIs) around the center of the wall and within unaffected muscle tissue (three ROIs) of the ipsilateral thigh (Fig. 1). The relative T*2-weighted SI values (SI of unaffected muscle − SI of abscess wall) were calculated and analyzed.
All animals were sacrificed by administration of inhalable pure CO2. The infected left calf was dissected and fixed in 10% buffered formalin. The pathologic specimens were embedded in paraffin blocks according to standard procedures. Sections 4 μm thick were cut transversely and stained with hematoxylin-eosin for histologic examination. The histologic findings were compared MR images. Prussian blue staining was performed to detect the presence of iron oxide particles.
Differences in the relative SI between the antagonist and control group of mice were assessed using the analysis of variance test and Student-Newmann-Keuls test. P values less than 0.05 were considered statistically significant.
MCP-1 Binding Affinity to CCR2
When RAW 264.7 cells were incubated with mMCP-1, a dose-dependent and saturable binding of mMCP-1 was seen. The equilibrium binding constant of mMCP-1 to RAW 294.7 cells was significantly decreased by the CCR2 antagonist (RS-102895) from 0.64 ± 0.07 nM to 0.34 ± 0.09 nM. The intensity of fluorescence measured at 5 nM of mMCP-1 was decreased by the CCR2 antagonist (MFI; control group = 564.7779 ± 51.62, 20-mM CCR2 antagonist group = 196.3126 ± 81.0) (Fig. 2). Although RS-102895 is developed as the human CCR2 antagonist, it can inhibit the binding affinity of mMCP-1 to the CCR2 of RAW 264.7 cells.
Reduced Expression of CCR2 and CD11b
Reverse transcription–PCR showed that the application of mMCP-1 to macrophages significantly increased the mRNA expression for CCR2, MCP-1, and CD11b in RAW 264.7 cells. Treatment with the CCR2 antagonist significantly reduced the increased expression of mRNA for CCR2, MCP-1, and CD11b caused by the mMCP-1 application (Fig. 3).
Flow cytometry used to investigate the effect of mMCP-1 on the expression of CCR2, showed that 10 ng/mL of mMCP-1 increased the CCR2 expression on the cellular surface (0 ng/mL mMCP-1 = 179.05, 10 ng/mL mMCP-1 = 244.27; relative MFI value). The up-regulation of CCR2 was significantly restricted by the CCR2 antagonist (10 ng/mL mMCP-1 = 235.75, 10 ng/mL MCP-1+CCR2 antagonist = 71.05; relative MFI value) (Fig. 4a). Similarly, the effect of mMCP-1 on CD11b of the cellular surface was inhibited by the CCR2 antagonist (0 ng/mL mMCP-1 = 4.2, 10 ng/mL mMCP-1 = 6.2; value of MFI) (Fig. 4a). Down-regulation of mouse CD11b protein by the CCR2 antagonist was also identified on confocal microscopy (Fig. 4b).
In Vitro Evaluation of the Effect of CCR2 Antagonist on Cell Migration
On the transwell migration assay, we found a significantly decreased number of macrophages treated with the CCR2 antagonist in the lower chamber with compared with macrophages treated with PBS. mMCP-1-induced chemoattractive migration of macrophages was limited by the CCR2 antagonist (Fig. 5a). The number of the cells treated with the CCR2 antagonist (18.7 ± 4.00 cells) was less than that of the control cells (50.4 ± 5.10 cells) in the lower chamber (without mMCP-1 application). The difference between the number of the cells treated with the CCR2 antagonist (52.8 ± 21.57 cells) and that of the control cells (238.0 ± 73.84 cells) was greater after the mMCP-1 application (Fig. 5b).
In Vivo MRI and Histologic Analysis
The abscess area showed slightly increased SI on T*2-weighted gradient-echo images. After administration of iron-oxide-labeled macrophages, a band-shaped, lower SI zone was noted in the abscess wall. The band-shaped lower SI zone was formed by the recruited macrophages. The SI of the band-shaped lower SI zone was significantly different in three groups (P < 0.05) (Fig. 6). The relative SI was significantly higher in the negative control group (mean relative SI = 9345) than in antagonist group (mean relative SI = 7168, P < 0.05). However, in the positive control group the relative SI (mean relative SI = −447) was lower than in the antagonist group. These findings indicate that recruited macrophages to the abscess wall were significantly decreased in the antagonist group compared with the negative control group, indicating that MCP-1-induced chemoattractive migration of macrophages to the infection site was reduced by the CCR2 antagonist.
In all mice, hematoxylin-eosin stain showed a cellular infiltrate of neutrophils and macrophages around the abscess wall between necrosis and intact muscles. Prussian blue stain was positive in macrophages of the cellular infiltrates that corresponded to areas with low SI on T*2-weighted gradient-echo images. The iron-oxide-labeled macrophages in abscess wall were decreased in the antagonist group more than in the negative control group (Fig. 7), and this finding was correlated with the findings of MRI.
In this study, we described CCR2 antagonist blocks the CCR2 of macrophages and decreases the chemotactic migration of macrophages toward MCP-1. Our data also show that the CCR2 antagonist decreases the recruitment of macrophages through the down-regulation of CD11b, as well as CCR2. Using in vivo MRI, the effect of the CCR2 antagonist on macrophage migration toward an abscess was successfully evaluated in this study.
CCR2 is a chemokine receptor predominantly expressed in monocytes and macrophages; this is important for mediating their tissue influx in the context of immune-based inflammation. CCR2 is a G-protein-coupled receptor, the ligands for which include the MCP family of chemokines. MCP-1 is present in high concentrations in various inflammatory lesions and is an important chemoattractive signal for monocytes and macrophages in inflammation (17, 18).
RS-102895 is a small-molecule chemokine receptor antagonist that is potent and specific for the CCR2b receptor. It is well known that the compounds block MCP-1 and MCP-3 signaling through CCR2; however, this does not affect CXCR1, CCR1, or CCR3. This antagonist blocks the receptor by occupation of a binding site (3). Our study showed that the CCR2 antagonist attenuated CCR2 expression and the migration of RAW 264.7 cells.
Many recent reports clearly demonstrate that the CD11b expression is not only a marker for inflammatory monocyte/macrophages but also directly regulates the process of monocyte recruitment (19–21). CD11b-dependent firm adhesion was required for migration of monocytes in response to MCP-1 (22). The up-regulation and adhesive function of CD11b are closely connected with the downstream of the CCR2 signal pathway (23). In our experiments, the MCP-1-induced up-regulation of CD11b was suppressed by the CCR2 antagonist.
Administration of CCR2 antagonists or neutralizing antibodies for its ligand, MCP-1, reduces inflammation in animal models, including adjuvant arthritis, lung granuloma, and glomerulonephritis (8–10, 17). Animals with genetically deleted CCR2 or MCP-1 are protected from inflammation and atherosclerosis induced by bacterial products and by high-fat diets (4, 24–26). Although these results clearly implicate CCR2 in the pathogenesis of macrophage-mediated inflammatory disorders, studies in knockout mice are inherently limited by their inability to exclude the impact of constitutive loss of CCR2 during development. Therefore, we believe it is important to demonstrate the effect of the CCR2 antagonist on macrophage migration in mice.
In our study, macrophages treated with the CCR2 antagonist showed a significant decrease of chemotactic migration toward MCP-1 in vitro. In MRI, recruited macrophages to the abscess wall were significantly decreased in the CCR2 antagonist group, more so than in the negative control group; this indicates that MCP-1-induced, chemotactic migration of macrophages to the infection site was inhibited by the CCR2 antagonist. Also in our study, macrophages treated with pertussis toxin did not show chemotactic migration to the infection site. These results were consistent with those of a previous report (27), thus suggesting that the action of MCP-1 is mediated by a G-protein-coupled receptor. Pertussis toxin is a protein-based, AB5-type exotoxin produced by Bordetella pertussis. Pertussis toxin catalyzes the ADP-ribosylation of the α subunits of the heterotrimeric guanine nucleotide regulatory proteins Gi, Go, and Gt and prevents intracellular signal transduction by preventing the G proteins from interacting with G-protein-coupled receptors on the cell membrane (28). Consequently, the relative SI of the CCR2 antagonist group was between that of the negative control and the pertussis toxin group. This indicates that several processes may mediate the chemotactic migration of macrophages to the infection site, other than the CCR2/MCP-1 system.
Our study had several drawbacks. First, we used RAW 264.7 cell lines, rather than monocytes, to study the effect of the CCR2 antagonist on the MCP-1/CCR2 system. It would be more physiologic if mouse monocytes were used in these experiments; however, to obtain monocytes from mice, we had to sacrifice too many mice. Therefore, we chose a monocyte/macrophage cell line that is similar to monocytes. Second, the degree of abscess could not be the same in all 12 mice, although the amount of inoculated Staphylococcus aureus and the duration for the development of abscesses were controlled in all 12 mice. Therefore, we assumed that the degree of abscess in the 12 mice did not differ significantly. Third, it could be an important limitation that human CCR2 antagonist was used for blocking the CCR2 on the mouse monocyte/macrophages. However, in our test for the change of binding affinity by human CCR2 antagonist, RS-102895, to murine CCR2, the CCR2 antagonist effectively bound and blocked murine CCR2 and decreased the migration of RAW 264.7 cells.
In conclusion, in vivo MRI successfully demonstrates the effect of the CCR2 antagonist on the macrophage migration toward muscle infection sites. CCR2 antagonist can block CCR2 on macrophages and can decrease the chemotactic activities of macrophages toward mMCP-1. Therefore, decreased expression of CD11b accompanied by decreased CCR2 may have an important role in the reduced recruitment of macrophages.