* Departments of Oncology, Osaka University Graduate School of Medicine, and
Toxicology and
Bioorganic Chemistry, Graduate School of Pharmaceutical Sciences, Osaka University, Japan; and
Department of Immunology, Research Institute, Osaka Medical Center for Maternal and Child Health, Japan
Correspondence: Dr. Hiromi Fujiwara, Department of Oncology, Osaka University Graduate School of Medicine, 2-2, Yamada-oka, Suita, Osaka 565-0871, Japan. E-mail: hf{at}ongene.med.osaka-u.ac.jp
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: chemokines • chemotaxis • CCR5 • CXCR3
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Within the T cell lineage, the expression of a given chemokine receptor appears to be restricted to particular groups of activated and/or memory T cells [8 , 9 ]. Many recent studies have shown that chemokine receptors are differentially expressed on antigen-stimulated T cells depending on their polarization [10 11 12 ], with T helper cell type 1 (Th1) lymphocytes expressing CCR5 and CXCR3 and Th2 lymphocytes CCR3, CCR4, and CCR8. Of these, Th1 cells are recruited to sites of inflammation and function as a key member to mediate inflammatory responses. In fact, T cells recovered from inflammatory sites such as the synovial fluid of rheumatoid arthritis (RA) have been shown to express CCR5 and CXCR3 [13 14 15 ]. Although these observations implied a role for CCR5 and CXCR3 in the migration of T cells to joint lesions, it is not evident whether they actually function to promote the process of migration. In this context, we have recently observed that administration of a nonpeptide, synthetic CCR5 antagonist, TAK-779 {N, N-dimethyl-N-[4-[[[2-(4-methylphenyl)-6, 7-dihydro-5H-benzocyclohepten-8-yl]carbon-yl]amino]benzyl]-tetrahydro-2H-pyran-4-aminium chloride; relative molecular weight=531.13}, along with collagen sensitization in the mouse RA model [16 , 17 ] inhibits the development of arthritis [18 ]. Although the results showed that a CCR5 antagonist exerted its effect by down-regulating T cell migration, the question of whether the migration is regulated only via CCR5 function under conditions in which the cells coexpress CXCR3 remains to be answered.
After establishing transfectants expressing mouse (m)CCR5, mCXCR3, mCCR4, or mCXCR4, the present study investigated the target receptor specificity of TAK-779. The results show that TAK-779 blocks the ligand binding of mCCR5 and mCXCR3 as well as mCCR5 or mCXCR3-mediated cell adhesion and chemotaxis but does not interfere with the ligand binding and function of mCCR4 and mCXCR4. Thus, this unique specificity of TAK-779 could provide a mechanistic explanation for the efficient blockade of Th1 migration to inflammatory lesions.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(mMIP-1), regulated on activation, normal T expressed and secreted (mRANTES), mMIP-1ß, m-IFN-inducible T cell alpha chemoattractant (mI-TAC), m-monokine induced by interferon-
(m-MIG), macrophage-derived chemokine (mMDC), and stromal cell-derived factor-1 (mSDF-1; R & D Systems, Minneapolis, MN); IFN-inducible protein 10 (mIP-10; PeproTech EC Ltd., London, UK); phycoerythrin (PE)-conjugated anti-mCCR5 monoclonal Ab (mAb; PharMingen, San Diego, CA); rabbit anti-mCXCR3 Ab (Zymed Laboratories Inc., South San Francisco, CA); biotinylated goat anti-rabbit immunoglobulin G (IgG; Jackson ImmunoResearch, West Grove, PA); rabbit anti-mCCR4 Ab (Alexis Biochemicals, San Diego, CA); goat anti-mMIP-1 Ab and goat anti-mMDC Ab (R & D Systems); goat anti-mIP-10 Ab and goat anti-mSDF-1 Ab (Santa Cruz Biotechnology, Inc., Santa Cruz, CA); fluorescein isothiocyanate (FITC)- and PE-conjugated streptavidin (Becton Dickinson, Mountain View, CA); FITC-conjugated donkey anti-goat IgG (Chemicon Inc., Temecula, CA); control rat IgG and rabbit IgG (Biomeda, Foster City, CA); and control goat Ig (R & D Systems). [125I]-Labeled human RANTES (hRANTES), [125I]hIP-10, [125I]-hMDC, and [125I]-hSDF-1 were products of Amersham Pharmacia (Buckinghamshire, UK).
Immunofluorescence staining and flow cytometry
mCCR5 was stained directly with PE-conjugated anti-mCCR5 mAb. mCXCR3 and mCCR4 were stained by incubation with rabbit anti-mCXCR3 or anti-mCCR4 Ab followed by biotinylated goat anti-rabbit IgG and PE- or FITC-conjugated streptavidin. mCCR5, mCXCR3, mCCR4, and mCXCR4 were also detected as the binding receptor for mMIP-1, mIP-10, mMDC, and mSDF-1, respectively, as described previously [20
]. Briefly, cells were incubated with the relevant chemokine (0.5 µM mMIP-1, mMDC, or mSDF-1 or 1 µM mIP-10) for 30 min at 4°C. After being washed, cells were allowed to react with the corresponding goat anti-mouse chemokine Ab and were then incubated with FITC-conjugated donkey anti-goat Ig. Stained cells were analyzed with a FACSCalibur (Becton Dickinson).
cDNA preparation
mCCR5 cDNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) from RNA isolated from the mouse T cell line 2D6 [21
]. mCXCR3 and mCCR4 cDNA were amplified from RNA of unfractionated spleen cells that had been treated with concanavalin A followed by restimulation with interleukin-2. The PCR products were then subcloned into pGEM-T Easy vector (Promega Corp., Madison, WI) and were completely sequenced to verify the correct reading frame and lack of any mutations. The mCXCR4-open reading frame (ORF) vector was prepared in a previous study [22
].
Retroviral DNA constructs
The retroviral vector used was derived by fusing the PclMFG-Lac Z vector (Retro Max System, Imgenex, San Diego, CA) and pRev-TRE vector (Clontech Laboratories, Inc., Palo Alto, CA). We first amplified a sequence including the cytomegalovirus (CMV) promoter, 5'-long terminal repeat (LTR) and the large packaging signal from the PclMFG-Lac Z vector by using the following primers: 5'-TGGGA-TCCGGCAGTCTAGAGGATGGTCCAC-3' and 5'-GGGAATTCTAGTTATTAATAGTAATCAATT-3'. The PCR products were then digested with EcoRI and BamHI and ligated to multiple cloning sites (MCS)- and 3'-LTR-containing fragment, which was derived by digesting the pRev-TRE vector with EcoRI and BamHI. The resulting fusion vector was named PclMFG X. The unique sites for SalI and SphI on pGEM-T Easy and PclMFG X vectors allowed us to directionally clone mCCR5 and mCXCR3, yielding PclMFG X–mCCR5 and PclMFG X–mCXCR3, respectively. To clone mCCR4, the insert in the pGEM-T Easy vector was excised with BamHI and HindIII, whose unique sites were contained in our design of the primers and were engineered to generate the retroviral vector PclMFG X–mCCR4. For engineering PclMFG X–mCXCR4, the mCXCR4–ORF was first digested with SalI, blunt-ended with T4 DNA polymerase, digested with NotI, and then ligated to a NotI–SphI-digested PclMFG X vector.
Transfection and infection
The 293T cells were used as packaging cells and transfected with retroviral vectors using the calcium phosphate method as described previously [23
]. The empty retroviral vectors were also transfected as a control. After 24 h, supernatants were replaced with complete Dulbecco’s modified Eagle’s medium, and the virus was allowed to accumulate for an additional 24 h. Viral supernatants were then collected, filtered, and mixed with 2B4 T cell hybridoma cells at a density of 1 x 105 cells/ml. Cells were spun at room temperature for 2 h at 2500 rpm and were incubated at 37°C for 48 h. Then, the surface expression of transfected genes was monitored by flow cytometry. Positive clones were selected by limiting dilution.
Preparation of the recombinant intercellular adhesion molecule-1 (ICAM-1)-Ig fusion protein
A genetic fusion construct encoding the first two Ig domains of mICAM-1 (D1D2: 1–185 amino acids) and the Cr1 domain of hIg were constructed by replacing the mouse cytotoxic T-lymphocyte antigen (mCTLA)-4 gene fragment of the recombinant CTLA-4–Ig chimeric DNA [24
] with the D1D2 ICAM-1 DNA fragment. The D1D2 fragment of ICAM-1 DNA was amplified by RT-PCR using the following primers: 5'-GAATTCTAGCACTTTGCCCTGGCC-3' and 5'-GGATCCAGGTCCATGGTGTCGAGCTTT-3'. The PCR products were digested with EcoRI and BamHI and were then inserted into a pSG5 vector containing a DNA fragment encoding the Cr1 domain of hIg, which was derived by digesting the pSG5–CTLA4–Ig chimeric DNA with EcoRI and BamHI as well. The resulting pSG5–ICAM-1–Ig chimeric DNA was transfected into COS 7 cells by electroporation using Gene Pulser (Bio-Rad, Richmond, VA) with 950 µF at 220 V. Chimeric fusion proteins were purified from culture supernatants using protein A-Sepharose 4 Fast Flow (Pharmacia, Uppsala, Sweden).
[125I]-Labeled chemokine-binding assay
A radiolabeled chemokine-binding assay was performed by referring to the previous papers [19
, 25
]. Briefly, 2B4 chemokine receptor transfectants (2x105/well) suspended in the binding buffer [50 mM HEPES, 1 mM CaCl2, 5 mM MgCl2, 0.5% bovine serum albumin (pH 7.4)] were incubated with [125I]-labeled relevant chemokines (specific activity: 2000 Ci/mmol) in 96-well microculture plates for 40 min at room temperature. The binding reaction was terminated by washing out the free ligand with cold phosphate-buffered saline (PBS), and the cell-associated radioactivity was measured by scintillation counter.
Cell adhesion assay
ICAM-1–Ig fusion protein (20 µg/ml in PBS, 40 µl/well) was coated overnight onto 96-well microculture plates (Corning 25860, Corning Glass Works, Corning NY). Plates were blocked by treatment with 1% ovalbumin at 37°C for 1 h. Cells were harvested, washed with PBS, and labeled with 5, 6-carboxy fluorescein diacetate succimidyl ester (10 µM in PBS) at 37°C for 15 min. Then, cells were washed and resuspended in adhesion buffer (Hanks’ balanced salt solution containing 10 mM HEPES, 2 mM MgCl2, and 1 mM CaCl2). When the antagonist TAK-779 was used, labeled cells were preincubated with different concentrations of TAK-779 in DMSO for 30 min on ice. After being washed, the cells (1x105 in 25 µl) were added to each well of ICAM-1-coated plates containing chemokines in 25 µl adhesion buffer. Plates were immediately centrifuged at 500 rpm at room temperature and incubated for 5 min at 37°C. Then, 2.5% glutaraldehyde in PBS (200 µl) was added to stop the reaction and fix the attached cells. Nonattached cells were removed by flicking off the solution, followed by washing with PBS three times. Then, 100 µl PBS was added, and the fluorescence of plate-attached cells was measured using the fluorescence concentration analyzer BioluminTM960 (Molecular Dynamics, Inc., Sunnyvale, CA). After the subtraction of background binding, specific binding in each group was expressed as the ratio to a chemokine-unstimulated control group (100%). Data are shown as the mean ± SE of three wells/group.
Chemotaxis assay
Chemotaxis was assessed in 24-well transwells equipped with 5 µM pore polycarbonate membranes (Transwell, Corning Costar Corp., Cambridge, MA). Cells (1x105) suspended in 100 µl RPMI-1640 medium supplemented with 10% fetal calf serum were transferred to the upper chamber. The lower chamber contained 600 µl medium with various concentrations of chemokines. After 2 h, unless otherwise indicated, the upper chambers were removed, and the lower chambers were left for 10 min to allow most cells to sink to the bottom. The cells that had migrated to the lower chambers were counted in five randomly selected fields under a microscope (200-fold magnification) as described previously [26
]. All assays were done in triplicate, and the results were expressed as number of cells/field (the mean±SE). Spontaneous migration was determined in the absence of chemoattractant.
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Figure 1. Establishment of mCCR5, mCXCR3, mCCR4, and mCXCR4 transfectants. Transfectants expressing mCCR5, mCXCR3, mCCR4, or mCXCR4 were established as described in Materials and Methods. Each transfectant or mock-transfectant was stained with antichemokine receptor Ab (A) or was incubated with the indicated chemokines and then stained with the corresponding antichemokine Ab (B).
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Figure 2. Chemokine dose responses of cell adhesion and chemotaxis in mCCR5 and mCXCR3 transfectants. mCCR5 and mCXCR3 transfectants were stimulated with various concentrations of chemokines in cell-adhesion (upper panels) and chemotaxis assays (lower panels). Data are representative of four similar experiments.
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instead. There exists very high homology or similarity of chemokine genes between the human and mouse species, with RANTES at 85%, IP-10 at 77%, MDC at 83%, and SDF-1 at 99%. Accordingly, several reports have already verified that human chemokines bind with high affinity to corresponding mouse chemokine receptors [28
29
30
], and we confirmed this using the transfectants developed in this study (data not shown). When the transfectants were preincubated with TAK-779, the antagonist efficiently inhibited the binding of [125I]-hRANTES and [125I]-hIP-10 to mCCR5 and mCXCR3 transfectants, respectively [50% inhibitory concentration (IC50): 236 nM for mCCR5 and 369 nM for mCXCR3], whereas it did not affect the capacity of mCCR4 to bind [125I]-hMDC or of mCXCR4 to bind [125I]-hSDF-1 (Fig. 3
).
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Figure 3. Inhibitory effect of TAK-779 on the binding of [125I]-labeled human chemokines to 2B4 cells expressing the relevant chemokine receptors. mCCR5, mCXCR3, mCCR4, and mCXCR4 transfectants were preincubated with various concentrations of TAK-779 for 30 min and were then incubated with [125I]-labeled hRANTES (0.5 nM), hIP-10 (0.5 nM), hMDC (1 nM), or hSDF-1 (0.25 nM), respectively, for an additional 40 min at room termperature. The percent-binding was calculated by the following equation: 100 x [(binding with inhibitor–nonspecific binding)/(binding without inhibitor–nonspecific binding)]. Nonspecific binding was determined in the presence of 300 nM unlabeled ligands. The amounts of radiolabeled ligands were determined based on the optimal signal to noise (nonspecific binding) ratios obtained in the preliminary experiments. The binding without inhibitor (cpm) and nonspecific binding (cpm) in each transfectant were 22,073 and 1282 (mCCR5), 33,793 and 1566 (mCXCR3), 11,480 and 935 (mCCR4), and 32,918 and 2215 (mCXCR4), respectively. Data are representative of two similar experiments.
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and mIP-10, respectively, whereas it did not affect the capacity of mCCR4 to bind mMDC or of mCXCR4 to bind mSDF-1.
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Figure 4. TAK-779 inhibits the interaction of mCCR5 and mCXCR3 but not of mCCR4 and mCXCR4 with the relevant chemokines. Each transfectant was preincubated with 1 µM TAK-779 on ice for 30 min. After being washed, mCCR5, mCXCR3, mCCR4, and mCXCR4 transfectants were incubated with 0.5 µM mMIP-1, 1 µM mIP-10, 0.5 µM mMDC, and 1 µM mSDF-1 at 4°C for 30 min, respectively, and were then stained with the relevant anti-mouse chemokine Ab. Data are representative of three similar experiments.
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Figure 5. TAK-779 inhibits cell adhesion and chemotaxis mediated by mCCR5 and mCXCR3 but not by mCCR4 and mCXCR4. Each transfectant was preincubated with different concentrations of TAK-779 on ice for 30 min before cell adhesion (upper panels) and chemotaxis (lower panels) and was stimulated for 5 min (cell adhesion assays) or 2 h (chemotaxis assays) with the following concentrations of chemokines: 10 nM mRANTES (mCCR5), 10 nM mIP-10 (mCXCR3), 10 nM mMDC (mCCR4), or 10 nM mSDF-1 (mCXCR4) for cell adhesion assays and 1 nM mRANTES (mCCR5), 10 nM mIP-10 (mCXCR3), 10 nM mMDC (mCCR4), or 10 nM mSDF-1 (mCXCR4) for chemotaxis assays. Adhesion and chemotaxis in the presence of antagonist were expressed as ratios to a medium control (DMSO/antagonist-free). Data are representative of four similar experiments.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Baba et al. [19
] developed TAK-779 as a CCR5 antagonist and reported that this antagonist does not react with other hCCR chemokine receptors except for hCCR2b, which exhibited detectable but weak levels of TAK-779 reactivity (
20-fold higher IC50 than that for hCCR5). Consistent with this, TAK-779 did not block mCCR4 in this study. The study of Baba et al. [19
], however, did not show its reactivity to receptors among another major chemokine receptor family CXCR. Our present study showed that TAK-779 exhibits comparable levels of blocking effects on mCXCR3 with those on mCCR5, and another CXCR, mCXCR4, is only marginally affected. As the IC50 values for mCCR5 and mCXCR3 were 236 nM and 369 nM, the difference between these (less than twofold) was apparently smaller than that (20-fold) observed between hCCR5 and hCCR2b [19
]. The finding that a CCR5 antagonist reacts selectively with another Th1 chemokine receptor, CXCR3, is quite interesting from a biological respect.
As mentioned above, TAK-779 exerted its inhibitory effects on the binding of mCCR5 and mCXCR3 to the relevant chemokines. However, the reactivity of this compound observed here in the mouse chemokine system greatly differed from that reported for the human system [19
]. In fact, the IC50 value for mCCR5 was 100-fold higher than that for hCCR5. Nevertheless, it should be noted that TAK-779 is effective at inhibiting T cell migration in vivo in the mouse models [18
, 31
]. Particularly in one of these studies [18
], TAK-779 did not produce any inhibitory effect on the generation of anticollagen T cell responses leading to Th1 chemokine receptor expression but inhibited the development of arthritis by modulating T cell migration to joints. Thus, despite the reduced TAK-779 reactivity in the mouse system, the effect of TAK-779 on mouse Th1 chemokine receptors has biological relevance.
The results obtained here may also provide additional information regarding the biology of chemokines and chemokine receptors apart from the action of the antagonist. In prior studies [28 , 32 33 34 35 36 37 38 39 ], the stimulation of CCR5 and CXCR3 was achieved with various concentrations of the respective ligands. As the experiments were done separately using different cell types expressing CCR5 or CXCR3, concentrations of ligands required for the optimal CCR5 and CXCR3 stimulation could not be compared properly. In this study, mCCR5 and mCXCR3 exhibited almost the same concentrations of ligand requirement for inducing lymphocyte function-associated antigen-1 (LFA-1) activation. In contrast, the chemokine dose requirement for inducing the optimal level of chemotaxis was more than one order of magnitude lower in mCCR5 than in mCXCR3. Thus, the present study, using the same cell type of transfectants, defines similar or differential chemokine-dose requirements for LFA-1 activation or chemotaxis, respectively, mediated by CCR5 and CXCR3.
If chemokines capable of stimulating CCR5 or CXCR3 are produced in inflammatory tissues at similar concentrations, the chemoattractant capacity would be one order of magnitude higher in CCR5 than CXCR3. However, such may not always be the case in inflammatory responses. Various chemokines have been detected at sites of inflammation (reviewed in ref. [40
]). A few studies [41
, 42
] compared the levels of CCR5- and CXCR3-ligand production. The studies that assessed chemokine expression in allografts [41
, 42
] showed that the expression levels of the CCR5-related chemokines RANTES, MIP-1, and MIP-1ß are significantly higher than those of CXCR3-related chemokines such as IP-10. Nevertheless, they [41
, 42
] also reported that CXCR3 and its ligand, particularly IP-10, play a key role in T cell recruitment and allograft rejection. The results showed that IP-10 is the first chemokine detected after allografting, and endothelial IP-10 expression in grafts promotes the initial recruitment of CXCR3+ natural killer and T cells, which serve to amplify and extend chemokine/cytokine production leading to enhanced allograft responses [41
]. Thus, it is possible that chemokine/chemokine receptor redundancy does not necessarily hold true for the CXCR3 and CCR5 pathways and instead, either pathway predominantly functions somewhere during the entire process of the inflammatory response. Overall, it is highly conceivable that the simultaneous blockade of CCR5 and CXCR3 results in efficient regulation of inflammatory responses. In this context, it is of significance that TAK-779, developed as a CCR5-specific antagonist, also interacts selectively with CXCR3 without exhibiting reactivity to other CCR and CXCR receptors. This explains how efficiently this antagonist works to inhibit the development of collagen-induced arthritis [18
].
It has been proposed that the N-terminal and the second extracellular loop are responsible for the ligand binding and HIV-1 entry of hCCR5 [43 44 45 ]. According to this view, TAK-779, capable of interfering with HIV-1 replication, may have direct interaction with these domains or induce conformational changes of these domains after binding to different parts of hCCR5 [19 ]. By alanine-scanning mutagenesis of the transmembrane (TM) domains, a recent study by Dragic et al. [27 ] showed that the binding site for TAK-779 on hCCR5 was located near the surface of the receptor, within a cavity formed between helices 1, 2, 3, and 7. Alanine substitution of five residues within these helices (L33, Y37, W86, Y108, and T123) had a strong, inhibitory effect on the antiviral activity of TAK-779 [27 ]. Although CCR5 and CXCR3 belong to different subfamilies and interact with totally different arrays of chemokines, these receptors in the mouse species exhibit 32% identity and 51% similarity. The similarity is as high as 72% for TM helices 1, 2, 3, and 7; the residues L33, Y37, and W86 of CCR5 are identical; and Y108 and T123 of CCR5 are similar in CXCR3. Thus, the possibility may be raised that a similar cavity exists to allow TAK-779 to sit in CXCR3. However, the above conservation pattern is found in the aligned sequence of hCXCR4 and mCXCR4, which do not react with TAK-779 (ref. [19 ] and this study). Therefore, the exact binding site of TAK-779 remains to be determined.
The present results illustrate the selective blokade of two Th1 chemokine receptors, mCCR5 and mCXCR3, by a nonpeptide antagonist. Regarding the binding of TAK-779 to mCCR5 and mCXCR3, further studies will be required to investigate whether a similar TAK-779-binding pocket exists in mCCR5 and mCXCR3 and if so, to confirm that the occupancy of such a binding site leads to the blockade of mCCR5 and mCXCR3 interactions with the corresponding chemokines. The present observations could, thus, have significant implications for the structural inter-relationship between CCR5 and CXCR3 interactions with different agonists and an identical antagonist.
Received June 2, 2002; revised October 30, 2002; accepted November 1, 2002.
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