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í Plach
* Medical Policlinic,
Institute for Animal Physiology, and
Institute for Anatomy, University of Munich, Munich, Germany;
Institute of Molecular Genetics, Czech Academy of Sciences, Prague, Czech Republic; and
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GSF National Research Center for Environment and Health, Institute of Molecular Immunology, Munich, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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5-fold in the progression phase, while CCR2 expression on other leukocyte populations remains unchanged. These findings identify CCR2+ T cells as regulatory T cells and indicate that CCR2 also plays an important role in down-modulating an inflammatory response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1% of the human population. Murine collagen-induced arthritis shares many features with the human disease and is a well-established model for rheumatoid arthritis (1). The murine disease can be separated in two phases: an initiation phase, in which the mice are immunized with collagen, resulting in a collagen-specific humoral and cellular immune response, and a progression phase, in which inflammatory cells migrate into the joints and arthritis becomes clinically apparent. We assumed that the chemokine receptor CCR2 plays an important role in both disease phases. During the initiation phase, APCs must migrate from the site of immunization to the draining lymph nodes to induce an efficient T and B cell response against collagen. Data from CCR2-deficient mice indicate that the migration of APCs to the draining lymph nodes is dependent on CCR2 (2, 3). Also in the progression phase, CCR2 should be an interesting target, as the recruitment of monocytes to sites of inflammation is dependent on CCR2, as shown in the model of thioglycolate-induced peritonitis with CCR2-deficient mice (4) or with blocking mAbs against CCR2 (5). In arthritis, monocytes are mainly responsible for joint destruction and perpetuation of the inflammatory process (6). The CCR2 ligand monocyte chemoattractant protein-1 (MCP-1)3 together with other proinflammatory chemokines is up-regulated in human rheumatoid arthritis (7, 8, 9) and in rodent models of collagen-induced arthritis (10, 11). In agreement with this expression of chemokines, we and others have found an impressive enrichment of CCR2- and CCR5-positive T cells and monocytes in the inflamed joints of patients with arthritis (12, 13, 14).
In murine collagen-induced arthritis, the chemokine receptor antagonists Met-RANTES (15), TAK-779 (16), and AMD-3100 (17) reduced the severity of arthritis. Application of the human MCP-1 analog (MCP-1 8–64) in MRL-lpr lupus mice resulted in an improvement of adjuvans-induced arthritis (18). In rats, arthritis was improved with blocking Abs against the chemokines MCP-1, RANTES, and macrophage-inflammatory protein (MIP)-1
(19, 20, 21).
The important role of CCR2 in inflammatory diseases has mainly been demonstrated with CCR2-deficient mice that have a better outcome in various models of inflammation (22, 23, 24). In contrast, lack of CCR2 impairs the control of infectious diseases such as tuberculosis (25) and Leishmania major (3). However, there are also reports that the absence of CCR2 can worsen organ function in noninfectious inflammatory diseases, as shown in the models of nephrotoxic serum nephritis (26), Aspergillus-induced asthma (27), and acetaminophen-induced hepatitis (28).
In this regard, it is important to note that chemokines are also involved in the recruitment of regulatory T cells. In humans, ligands for the receptors CCR4 and CCR8 preferentially attract regulatory T cells (29). Ag-activated murine B cells secrete the chemokines MIP-1, MIP-1
, and MCP-1 that are able to recruit regulatory T cells in vitro and suppress activation of B cells (30). Regulatory CD4+ T cells are phenotypically defined by a set of surface markers, including CD25+ and CD103 (integrin E)+ (31, 32). Regulatory CD4+ T cells play an important role in maintaining peripheral tolerance. They are anergic to activation and actively suppress immune responses in a cell-cell contact-dependent manner (31, 33).
To date, no data are available on the outcome of blocking CCR2 in relevant animal models of rheumatoid arthritis. We have chosen the model of collagen-induced arthritis in DBA/1 mice and used a previously characterized blocking mAb against CCR2 (MC-21) compared with an isotype control Ab (MC-67) (5). The use of Abs enabled us to separately block CCR2 in the initiation phase (days 0–15) and progression phase (days 21–36) of the disease. We show that improvement of arthritis is only achieved when CCR2 is blocked during the initiation phase. In contrast, blockade of CCR2 during the progression phase markedly aggravates arthritis and increases the humoral immune response against collagen. We provide evidence that CCR2+ T cells belong to the subset of regulatory T cells and that their number markedly increases during disease progression. Our data suggest that interference of the CCR2 Ab with regulatory T cells is responsible for the increased arthritis in the progression phase.
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Materials and Methods |
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The CCR2 Ab MC-21 was shown to block murine CCR2 in vitro and in vivo with an IC50 below 0.1 µg/ml (5, 34). No significant depletion of CCR2+ T cells or monocytes was observed in the peripheral blood or spleens of DBA/1 mice treated with MC-21 (data not shown). The Ab MC-67 (rat IgG2b) was used as isotype control. Both Abs were purified by protein G-Sepharose. Where indicated, LPS was removed on Mono-S columns (Pharmacia, Peapack, NJ).
Induction and evaluation of collagen-induced arthritis
Bovine collagen type II (Sigma-Aldrich, St. Louis, MO) was dissolved overnight in 0.1 M acetic acid at a concentration of 2 mg/ml and emulsified in an equal volume of CFA for the first injection and IFA for the second immunization. Age-matched 8- to 12-wk-old male DBA/1 mice were immunized with an intrasubcutaneous injection of 100 µl emulsion at the tail base on days 0 and 21. Beginning from day 0 or 21, mice were treated six times every third day by i.p. injection of 500 µg CCR2 Ab (MC-21) or isotype control Ab (MC-67) in PBS. Three days after injection, the plasma MC-21 concentration measured by flow cytometry on CCR2-transfected Chinese hamster ovary cells was above 5 µg/ml, which is sufficient to completely block CCR2 (5). The clinical score of arthritis was evaluated by two independent investigators: 0, normal; 1, swelling in one joint; 2, swelling in more than one joint; 3, swelling of the entire paw; 4, deformity and/or ankylosis. Animal experiments were performed in accordance with the legal requirements of the Government of Bavaria (Az. 211-2531-25/2000).
Histology
Hind paws were fixed in 10% Formalin for 24 h and embedded in methylmethacrylate to obtain optimal articular histology. After cutting 5-µm-thick sections with a polycut (Leica, Bensheim, Germany), sections were stained with Giemsa and the following parameters were evaluated on a scale from 0 (normal) to 2 on at least 10 sections of the tarsometatarsal joints by two independent investigators in a blinded fashion: synovial inflammation (1, focal inflammatory infiltrates; 2, inflammatory infiltrate dominating the cellular histology), synovial hyperplasia (1, continuous, at least three-layer-thick synovial lining in one joint; 2, in several joints), pannus formation and cartilage loss (1, cartilage partially covered by pannus, no cartilage loss; 2, with cartilage loss), and bone destruction (1, small areas of bone destruction; 2, widespread bone destruction).
FACS analysis and isolation of leukocytes from the joints
Leukocytes were isolated from the inflamed ankle joints by careful removal of the inflamed synovial tissue and subsequent digestion of the synovial tissue with 1 mg/ml collagenase type I (Sigma-Aldrich) for 20 min at 37°C. For FACS analysis, cells were stained with 5 µg/ml primary Abs MC-21 or MC-67, followed by a biotinylated polyclonal rabbit anti-rat Ab (DAKO, Carpenteria, CA). After blockade with 10% rat serum, streptavidin-PE or -Cy5 and a selection of the following directly labeled Abs were added: CD4 allophycocyanin, CD8 CyChrome, CD11b FITC, CD44 FITC, CD44 PE, CD44 CyChrome, CD45RB PE, CD19 PE, CD25 FITC, CD25 PE, CD103 PE, and GR-1 allophycocyanin (all BD PharMingen, San Diego, CA). Where indicated, CCR2 was down-modulated before staining by incubation for 30 min at 37°C with 1 µg/ml of murine MCP-1/JE. Cells were analyzed with a FACSCalibur (BD Biosciences, San Diego, CA).
In vitro restimulation of splenocytes with collagen
Splenocytes (5 x 106/ml) were cultivated for 48–72 h in the presence or absence of bovine collagen type II (40 µg/ml) in RPMI 1640 medium with 10% FCS. Where indicated, the Abs MC-21 or MC-67 were added (50 µg/ml). To measure cell proliferation, splenocytes were stained for 15 min at 37°C with 5 µM CFSE in PBS/0.1% BSA and stimulated for 72 h with collagen, as described above. Cells were stained with a combination of Abs (CD25 PE, CD44 CyChrome, CD4 allophycocyanin and CD19 PE, CD44 CyChrome, and CD8 allophycocyanin for Fig. 3b; CD19 PE, CD8 CyChrome, and CD4 allophycocyanin for Fig. 6b; and CD44 PE, CCR2 CyChrome, and CD4 allophycocyanin for Fig. 9) and analyzed by flow cytometry. After gating on the lymphocyte population with careful inclusion of larger activated lymphocytes, subpopulations were identified according to their expression of CD19, CD4, CD8, CD44, and CCR2. Proliferating cells were identified by reduced staining for CFSE. The percentage of proliferating cells was calculated for each lymphocyte subpopulation as: (number of CFSElow cells)/(number of CFSEhigh cells + number of CFSElow cells) x 100.
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ELISA
IFN-
and IL-4 (OptEIA; BD PharMingen) and Abs against collagen were quantified by ELISA. Collagen (30 µg/ml) was coated overnight on ELISA plates. Plasma samples were applied in a dilution of 1/40,000 in PBS/1% BSA, followed by a HRP-labeled polyclonal rabbit anti-mouse Ab (P260; DAKO) or HRP-labeled mAbs specific for murine IgG1 (clone LO-MG1-2; Serotec, Oxford, U.K.) or for murine IgG2a (clone R19-15; BD PharMingen).
Cell preparation and proliferation assays
CD4+ T cells were isolated from the spleens of DBA/1 or BALB/c mice by negative selection using magnetic beads (Miltenyi Biotec, Gladbach, Germany) and further separated in CCR2- and CCR2+ cells by FACS using a modular flow cytometer (Moflo; DakoCytomation, Fort Collins, CO). The purity of CD4+ CCR2- and CD4+ CCR2+ T cells was 98 and 93%, respectively. CD19+ B cells were isolated with magnetic beads (Miltenyi Biotec) and reached a purity of 98%. CFSE-stained CCR2- CD4+ T cells (50,000) were activated for 3 days with 0.5 µg/ml anti-CD3 mAb (clone 145-2C11; BD Biosciences) together with 50,000 B cells as costimulatory cells in 96-well round-bottom plates. CCR2+ CD4+ T cells were added at various concentrations. Total volume was 200 µl/well. B cells were labeled with 3.3 µM CFSE, as described above. CFSE-stained B cells (80,000) and CCR2- CD4+ T cells (20,000) were incubated with LPS (50 µg/ml) and anti-CD3 mAb (0.5 µg/ml) for 3 days in 96-well round-bottom plates in RPMI 1640 with 15% FCS, 1 mM sodium pyruvate, 50 µM 2-ME, and nonessential amino acids. CCR2+ CD4+ or CCR2- CD4+ T cells were added at the concentration given in the figures. The supernatant was recovered, and cells were stained with CD4 APC and CD19 PE Abs and analyzed on a flow cytometer (FACSCalibur; BD Biosciences) using fluorescent beads to exactly determine the number of cell divisions per well. Cell divisions per well were calculated by 
(gi x ni) x (number of added beads)/(number of counted beads), with gi being the generation number (0 for nondivided cells), and ni being the number of cells in each generation. Experiments were performed in duplicates and repeated at least twice.
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Results |
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Blockade of CCR2 in the initiation phase (days 0–15) resulted in a significant improvement of the clinical score measuring joint inflammation (Fig. 1A). Mice treated with the CCR2 Ab reached a mean score of less than 2, while mice treated with the isotype control Ab showed a mean score of >5. This was paralleled by a significant improvement of histological scores obtained on the lower tarsometatarsal joints (Fig. 1, B and C). All four parameters (inflammatory infiltrate, bone and cartilage erosion, and synovial hyperplasia) were reduced by 50% in MC-21-treated animals.
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release in MC-21-treated animals (data not shown), the anti-collagen Ab titers were significantly reduced by the blockade of CCR2 (Fig. 2). Both total IgG and IgG2a levels of collagen-specific Abs were reduced in the anti-CCR2 (MC-21)-treated group at the end of the experiment (day 40) with no shift of Ab subclasses from IgG2a to IgG1.
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(Fig. 3A) and proliferation of T cells (Fig. 3B). Incubation of splenocytes with collagen in the presence of CCR2 mAbs (MC-21) significantly reduced the release of IFN- (Fig. 3A) as well as the percentage of proliferating T cells (Fig. 3B). This indicates that CCR2 also has a direct influence on the Ag-specific activation of T cells independent of cell migration. Blockade of CCR2 during progression of collagen-induced arthritis
In the second set of experiments, we analyzed the outcome of collagen-induced arthritis when CCR2 was blocked during the progression phase of the disease (days 21–36). Very surprisingly, blockade of CCR2 in the progression phase resulted in a marked aggravation of the clinical signs of arthritis (Fig. 4A) and a concomitant worsening of all histological parameters (Fig. 4B). Already after the fourth injection the clinical score was more than 2-fold higher in MC-21-treated mice with frequently observed deformation of joints. The histological scores for bone and cartilage erosion and synovial hyperplasia were about 2-fold higher in the MC-21 group than in the control group, and also the number of infiltrating leukocytes, including monocytes/macrophages, was significantly increased (p < 0.01) (Fig. 4B). We can exclude LPS contamination as explanation for the increased arthritis, as both Abs (MC-21 and MC-67) were always produced under the same conditions, and use of LPS-free Ab preparations (LPS below 0.5 ng/mg Ab) gave the same results. To determine whether the infiltrating cells still express CCR2, we prepared single cell suspensions of the inflamed tissue surrounding the joints and analyzed the infiltrating cells by flow cytometry. Approximately 50% of the infiltrating cells in the synovial tissue were monocytes, 40% neutrophils, and 10% T cells. Monocytes in the inflamed joints were homogeneously CCR2 positive, and 40% of the CD4+ T cells expressed CCR2 in both groups (Fig. 5). These data were confirmed by down-modulation of CCR2 with MCP-1/JE, demonstrating that CCR2 can be internalized on infiltrating cells (Fig. 5).
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2.5% of the B cells proliferated after activation with collagen, while less than 0.5% of the B cells responded toward collagen in the control group (Fig. 6B). No shift in the Ab subtypes from IgG2a to IgG1 was detectable. Collagen-specific T cell proliferation was not significantly different in both groups. The level of IL-4 release was below the detection limit of our ELISA (10 pg/ml) in both groups. CCR2-positive T cells are anergic and have potent suppressor activity
The marked aggravation of arthritis by blockade of CCR2 during disease progression suggests that CCR2-dependent counter-regulatory mechanisms are involved in the control of the immune response against collagen in the progression phase. Collagen-induced arthritis is a partially self-limiting disease with a maximum of arthritis at days 40–45. The appearance of regulatory T cells during the course of the disease might be one way to confine the inflammation. We therefore analyzed in detail the expression of CCR2 on various lymphocyte subpopulations. Although CCR2 was not detectable on B cells, we found that CCR2 is expressed on CD4+ regulatory T cells phenotypically defined by the surface markers CD45RBlow, CD44high, CD25+, and CD103+. CCR2 expression was only detectable in the subset of CD44high CD45RBlow T cells (Fig. 7A). CD25+ T cells could be separated in two populations according to their expression of CD44 and CCR2. One CD44high subpopulation is homogeneously CCR2 positive, while the CD44low subpopulation is CCR2 negative (Fig. 7B). Approximately 70% of the CD103+ CD4+ T cells express high amounts of CCR2 (Fig. 7C). In addition, we noted marked changes in the subpopulations of CCR2+ CD4+ T cells during the course of collagen-induced arthritis (Fig. 8). The percentage of CD25+ CCR2+ T cells increased 5-fold after the second immunization with collagen, while CD25- CCR2+ T cells were temporarily reduced after the first immunization.
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Discussion |
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Blockade of CCR2 during the initiation phase (days 0–15) significantly attenuated the clinical disease score and the histological signs of arthritis. It appears very unlikely that the blockade of CCR2 from days 0 to 15 directly affects leukocyte migration into the joint, as the majority of leukocyte influx takes place after day 26. No signs of joint inflammation are visible until day 26, and in clinically unaffected joints we never detected any histological signs of arthritis with inflammatory cells. We therefore investigated whether CCR2 blockade during disease initiation could reduce the immune response against collagen and thereby the severity of the disease. Indeed, this interpretation is supported by the significantly reduced plasma titers of anti-collagen Abs in MC-21-treated mice. The induction of collagen-specific Abs has been shown to be important for the development of arthritis and may at least partially explain the protective effect of the CCR2 blockade during disease induction (35, 36).
Two mechanisms may explain why the CCR2 blockade in the initiation phase affects the immune response against collagen. First, it has been described that migration of APCs from sites of immunization to draining lymph nodes is reduced in CCR2-deficient mice (2, 3). This would lead to a reduced immune response against injected Ags. Second, blockade of CCR2 could directly impair the ability of APCs (CCR2+ monoctyes) to activate T cells after challenge with Ag. To test the latter hypothesis, we restimulated splenocytes in vitro with collagen in the presence of CCR2-blocking Abs. Blockade of CCR2 significantly reduced the collagen-specific T cell activation. A direct effect of the CCR2 Ab on T cells appears very unlikely, as only CCR2-negative T cells proliferate in response to collagen (Fig. 9) and the suppressive activity of CCR2-positive T cells is not altered by the blockade of CCR2 in vitro (data not shown). Moreover, polyclonal T cell activation induced with CD3 Abs was not influenced by the blockade of CCR2 (data not shown). Similarly, no differences have been described between CCR2+/+ and CCR2-/- mice, when splenocytes were stimulated with CD3 and CD28 Abs (2). This shows that the inhibitory effect of CCR2 Abs on T cell activation is dependent on the stimulus used for T cell activation.
Surprisingly, blockade of CCR2 during progression of the disease (days 21–36) resulted in a severe aggravation of arthritis. This was especially unexpected, as we assumed that migration of monocytes into the joints might be partially dependent on CCR2. Monocytes/Macrophages constitute a large proportion of the cellular infiltrate in the joints and are homogeneously CCR2 positive. The increased migration of leukocytes into the joints after CCR2 blockade indicates that monocyte migration into the joints is not dependent on CCR2 and points to the contribution of other chemokines (e.g., CCR1 ligands) or inflammatory mediators (e.g., complement components). Furthermore, mice that were immunized only once with collagen and treated from days 21 to 36 with MC-21 also showed more severe disease (data not shown), indicating that the concomitance of the second immunization with the MC-21 treatment is not a critical point. We can also exclude LPS contamination as explanation for the increased arthritis, as both Abs (MC-21 and MC-67) were always produced under the same conditions, and use of LPS-free Ab preparations gave the same results.
Analyzing the humoral and cellular immune response against collagen in mice treated during disease progression, we found significantly higher collagen-specific Ab titers in the plasma of MC-21-treated animals and an increased percentage of B cells proliferating in response to collagen. Collagen-specific T cell responses were unchanged, and no release of IL-4 was detectable. A potential explanation for the increased B cell response in MC-21-treated animals could be a shift from Th1 to Th2. However, we did not find signs for a shift toward Th2, as no increased IL-4 release and no shift of Ab subtypes from IgG2a to IgG1 were detectable. In addition, a shift toward a Th2 response should also have resulted in an increased humoral immune response in the initiation phase, which clearly was not the case. We therefore evaluated the possibility that CCR2-dependent counter-regulatory mechanisms are involved in the control of the immune response against collagen in the progression phase of the disease. Collagen-induced arthritis is a partially self-limiting disease with a maximum of arthritis at days 40–45 under our experimental conditions and a decrease of the clinical score thereafter. This indicates that the inflammatory process can be controlled by the host. The appearance of regulatory T cells during the course of the disease might be one way to confine the inflammation. Recently, it was shown that activated murine B cells release the chemokines MIP-1, MIP-1, and MCP-1 that attract regulatory T cells and suppress B cell activation (30). We therefore analyzed in detail the expression of CCR2 on CD4+ T cell subpopulations and found that CCR2 is expressed on regulatory T cells phenotypically defined by the surface markers CD45RBlow, CD44high, CD25+, and CD103+. CCR2+ T cells also fulfill the functional criteria of regulatory T cells, namely anergy and active suppression of other T and B cells (30, 31). Polyclonal or collagen-specific stimulation of T cells resulted only in proliferation of CCR2- T cells, while the CCR2+ T cells were completely anergic. The suppressive activity of CCR2+ T cells was demonstrated by coincubation with CFSE-labeled CCR2- responder T cells in different ratios. CCR2+ T cells almost completely blocked the polyclonal and Ag-specific proliferation of responder T cells in a dose-dependent manner. CCR2+ T cells also efficiently suppressed the LPS-induced proliferation of B cells. However, it remains unclear whether the CCR2+ T cells directly affect B cells or whether they are primarily directed against Th cells that in our hands were necessary to obtain acceptable B cell proliferation. In contrast to CCR2+ T cells, the CCR2- T cells markedly enhanced B cell proliferation.
The aggravation of arthritis and the increased humoral immune response in mice treated with CCR2 Abs during disease progression might well be explained by the effect of CCR2 Abs on CCR2+ regulatory T cells arising during the course of arthritis. The number of CD25+ CCR2+ CD4+ T cells increases 5- to 6-fold during disease progression. Although CCR2+ regulatory T cells are anergic in vitro, they obviously can proliferate in vivo during disease progression. These data are in accordance with recent reports that demonstrate proliferation of regulatory CD25+ T cells in vivo after immunization (37). We assume that CCR2 is necessary to bring regulatory T cells to the proper place in vivo, e.g., in the proximity to B cells, and that the blockade of CCR2 interferes with this migration. The correct localization of regulatory T cells is important, as their suppressive activity is largely cell contact dependent (33). We have excluded two other possibilities as to how the CCR2 Ab could affect CCR2+ regulatory T cells. Ab treatment of mice in the progression phase did not result in depletion of CCR2-positive T cells, nor did we observe a direct inhibitory effect of CCR2 Abs on the suppressive activity of regulatory T cells in vitro (data not shown). In addition, we analyzed whether CCR2 is necessary for the development of regulatory T cells. Comparing CCR2-deficient mice and wild-type controls, we did not find a reduced frequency of CD25+ CD44high CD4+ T cells in CCR2-deficient mice.
In conclusion, blockade of CCR2 has completely opposite effects during initiation and progression of collagen-induced arthritis. Blockade of CCR2 is only beneficial during the initiation phase, while in the progression phase it leads to a marked aggravation of arthritis. In the progression phase, interference of the CCR2 Ab with the function of CCR2+ regulatory T cells appears to be responsible for the aggravation of arthritis. We observed marked changes in the number of CCR2+ T cells with a several-fold increase of CCR2+ CD25+ CD4+ T cells during disease progression, and demonstrate that CCR2+ T cells have potent suppressive activity against T and B cells. These data show that the proinflammatory chemokine receptor CCR2 also has anti-inflammatory properties and underline the importance of chemokines in immune regulation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Matthias Mack, Medical Policlinic, University of Munich, Pettenkoferstr. 8a, 80336 Munich, Germany. E-mail address: mack{at}medpoli.med.uni-muenchen.de
3 Abbreviations used in this paper: MCP, monocyte chemoattractant protein; MIP, macrophage-inflammatory protein.
Received for publication August 6, 2003. Accepted for publication November 4, 2003.
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T. H. Terwey, T. D. Kim, A. A. Kochman, V. M. Hubbard, S. Lu, J. L. Zakrzewski, T. Ramirez-Montagut, J. M. Eng, S. J. Muriglan, G. Heller, et al. CCR2 is required for CD8-induced graft-versus-host disease Blood, November 1, 2005; 106(9): 3322 - 3330. [Abstract] [Full Text] [PDF]
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T. Nanki, Y. Urasaki, T. Imai, M. Nishimura, K. Muramoto, T. Kubota, and N. Miyasaka Inhibition of Fractalkine Ameliorates Murine Collagen-Induced Arthritis J. Immunol., December 1, 2004; 173(11): 7010 - 7016. [Abstract] [Full Text] [PDF]
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A. E. Herman, G. J. Freeman, D. Mathis, and C. Benoist CD4+CD25+ T Regulatory Cells Dependent on ICOS Promote Regulation of Effector Cells in the Prediabetic Lesion J. Exp. Med., June 7, 2004; 199(11): 1479 - 1489. [Abstract] [Full Text] [PDF]
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) homogeneously express CCR2 (gray area), while neutrophils (PMN) are CCR2 negative (gray area) (lower panels). The specificity of the CCR2 staining was controlled by internalization of CCR2 with MCP-1/JE (1 µg/ml) for 30 min at 37°C (upper right panel and thick line in the lower panel) and by use of an isotype control (vertical lines in the upper panels; thin lines in the lower panels).
