Cloning and expression analysis of rabbit CCR2

Full-length cDNA sequence of rabbit CCR2 was cloned from total spleen RNA of New Zealand white rabbits using a PCR based method. The deduced amino acid sequence of rabbit CCR2 encoded a protein of 369 amino acids (Fig. 1) that shared 80 % identity to the human CCR2b receptor. The sequence alignment with the mouse, rat and rhesus monkey CCR2 is also depicted in Fig. 2. Rat and mouse CCR2 contained additional 13 amino acids at the N-terminus compared to human, monkey and rabbit CCR2. In addition, one amino acid deletion in rabbit CCR2 was observed at position 18 (Fig. 2). Cysteines that were essential for the disulfide bond formation were also conserved in rabbit CCR2. Based on the SS and 2SS model [16], cysteine pairs (Cys-72/Cys-119 and Cys-31/Cys-276) in rabbit CCR2 were highly conserved compared to its counterparts from all known species. Expression levels of rabbit CCR2 were also determined in various rabbit tissues using Taqman analysis. Rabbit CCR2 was abundantly expressed in lungs and spleen compared to low levels of expression in brain, heart, liver and testis (Fig. 3).

Radioligand binding studies in rabbit CCR2 expressing cells

Stable cell lines expressing rabbit CCR2 in U-937 cells were generated to characterize the binding properties of radiolabeled human MCP-1 or mouse JE to rabbit CCR2. The binding affinity of radiolabeled human MCP-1 or mouse JE was measured using a competition binding assay of the respective cold ligands. As shown in Figs. 4, ¹²⁵I-mouse JE showed high affinity with a calculated K _d of 0.95 ± 0.02 nM and B _max of 18829 ± 1555 dpm. In contrast, ¹²⁵I-human MCP-1 has less binding affinity to rabbit CCR2 with lack of saturable binding as high as 6 nM of hot ligand (data not shown). Detailed examination of the binding characteristics of radiolabeled human MCP-1 to rabbit CCR2 will be necessary to resolve this discrepancy. To address the point, future studies are necessary to understand rabbit CCR2 binding characteristics and pharmacology with ¹²⁵I-rabbit- MCP-1.

Receptor pharmacology of rabbit CCR2 was studied using a variety of ligands that belonged to the MCP subfamily and irrelevant ligands such as RANTES, MIP-1α and MIP-1β. As expected, human MCP-1, -2, and -4 differentially competed the binding of radiolabeled mouse JE with K _i values of 8.4, 13.5 and 0.44 nM, respectively. In contrast, cold MCP-3 failed to compete JE binding to rabbit CCR2 when used at concentrations as high as 30 nM (Fig. 5A). In addition, irrelevant ligands that bind with high affinity to CCR1 or CCR5 were unable to compete binding of radiolabeled mouse JE to rabbit CCR2, while cold JE competed binding with a Ki value of 0.116 nM (Fig. 5B). TAK-779 is a well characterized small molecule antagonist of both CCR2 and CCR5, and has a 27 nM affinity against human CCR2b receptor [17]. We tested the activity of TAK-779 on rabbit CCR2 transfected cells using radiolabeled mouse JE in a standard competition binding assay. As shown in Fig. 5C, TAK-779 was also highly potent against rabbit CCR2 with an IC₅₀ of 2.3 nM.

Functional characterization of Rabbit CCR2 stable cell line

In order to confirm whether rabbit CCR2 expressed in U-937 cells is functionally coupled to G_αi family of G proteins, chemotaxis assays were conducted using both parental U-937 and U-937/rabbit CCR2 stable transfectants. Cells were incubated in the top chamber in the presence of increasing concentrations of ligand in the lower chamber. Parental U-937 failed to migrate to either human MCP-1 or mouse JE. In contrast, U-937/rabbit CCR2 stable transfectants migrated effectively to either human MCP-1 or mouse JE (Fig. 6A and 6B). The maximal migration was observed at roughly 4 nM of ligand and ranged from 20–40 % total input compared to 0.5–1 % of cells migrating in the absence of any ligand. A typical bell-shaped curve was observed in the chemotaxis response with either human MCP-1 or mouse JE. As expected, both parental and rabbit CCR2 stable cell lines migrated well to the CXCR4 ligand SDF-1α (Fig. 6C). The antagonist effect of TAK-779 was also examined against rabbit CCR2 in the presence of defined concentration of either mouse JE or hMCP-1 (4 nM). Preincubation of rabbit CCR2 expressing cells with TAK-779 effectively blocked migration of either mouse JE or hMCP-1 with an IC₅₀ of 98 nM and 4 nM, respectively (Fig. 6D and 6E). As expected, TAK-779 inhibited MCP-1 dependent migration of U-937/hCCR2b stable cells with an IC₅₀ of 3.8 nM (Fig. 6F)

Materials

Recombinant human chemokines (MCP-1, -2, -3, -4, RANTES, MIP-1α, MIP-1β) were purchased from Pepro Tech (Rocky Hill, NJ). Mouse JE and human SDF-1α were obtained from R&D Systems (Minneapolis, MN). Radiolabeled ligands (¹²⁵I-human MCP-1 and ¹²⁵I-mouse JE) were purchased from Perkin Elmer (Boston, MA). RPMI 1640, bovine albumin serum (BSA) and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) were purchased from Sigma (Gaithersburgh, MD). Polymeric chain reaction (PCR) and Taqman primers were purchased from Qiagen (Valencia, CA). The CCR5/CCR2 dual antagonist TAK-779 was synthesized as described elsewhere [14].

Rabbit cDNA cloning

Total RNA was prepared from the spleen of New Zealand white rabbits by the Trizol method and the cDNA was transcribed by using SuperScript™ First-Strand Synthesis System for RT-PCR (Invitrogen, Carlson, CA). Then the cDNA was amplified by PCR by using conserved primers (primer 1: 5'-GTATTCATCTTTGGTTTTGTGGGCAACATG-3' and primer 2: 5'-CAAAGGTAACTGTCCTGGCTTTTAAAGCAA-3'). The resultant PCR fragment was sequenced and this information was used to design the rabbit CCR2 gene specific primers. Rabbit CCR2 gene specific primers were used to amplify two fragments of rabbit cDNA by 5'- and 3'- rapid amplification of cDNA ends (RACE) using SMART RACE cDNA Amplification Kit (Clonetech, Palo Alto, CA). The sequence information from 5'- and 3'- RACE products was further used to design two primers (5'-GGTTGCTGAGAAGCCTGACACGC-3', and 5'-CAGGTCTGTATTCTTCAACAAGCCCTCG-3') out of start and stop codons, and full-length rabbit CCR2 cDNA was cloned using PCR amplification. The final PCR product with corresponding size was cloned to PCRII-TAPO (Invitrogen) and sequenced.

Tissue distribution

Rabbit cDNA from brain, heart, liver, lung, spleen and testis were quantitated for expression levels of CCR2 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by Taqman method. TaqMan primers and probes were designed using the Primer Express Computer Program (Applied Biosystems, Foster City, CA). The forward and reverse primers and probe selected for rabbit CCR2 are: 5'-catgacacactgctgcatcaac-3', 5'-gagaggtagctccggaacttctc-3' and 5'-[6-FAM]-ccgtggtctacgccttcgtcgg-[TAMRA-6-FAM]-3'. The forward, reverse primers and probe selected for rabbit GAPDH are: 5'-ggatttggccgcattgg-3', 5'-caacatccactttgccagagttaa-3' and 5'-[6-FAM]-cgcctggtcaccagggctgct-[TAMRA-6-FAM]-3'. Amplification mixture contained a total volume of 10 μl with 6 μl of TaqMan universal PCR master mixture (300 nM forward primer, 300 nM reverse primer, and 900 nM probe) and 4 μl of diluted samples. Taqman amplification was conducted on a ABI PRISM 7900HT Sequence Detector System (Applied Biosystems, Foster City, CA) with the following thermal profile: 1 cycle each of 50°C for 2 minutes and 95°C for 10 minutes followed by 40 cycles each of 95°C for 15 seconds and 60°C for 1 minute. Data was analyzed using a built-in standard curve method.

Generation of retrovirus infected rabbit CCR2 stable cell line

Rabbit cDNA described above was amplified by PCR with primer 11: 5'-ggaggcagatct cgaacaggcagctggcgg-3' and primer 12: 5'-ttattggaattc caagccgacggctgtcca-3' (underlined nucleotides are the cleavage sites of Bgl II and EcoR I, respectively). The resulted PCR fragment was digested with Bgl II and EcoR I, and then subcloned into the corresponding site of pMSCV_puro retroviral expression vector (BD Biosciences, CA). Lipofectamine-mediated transfection was carried out in the AmphoPack 293 packaging cell line (BD Biosciences, CA) with a full-length cDNA for rabbit CCR2 in the retroviral expression vector pMSCV_puro, containing a puromycin resistance selection marker. Thirty six hours after transfection, the pool of viral supernatant was recovered from the packaging cells, centrifuged, and polybreen was added to a final concentration of 8 μg/ml. The viral supernatant was then added to parental U-937 cells in a tissue culture treated flask and the infection was allowed to proceed for 7 hours. At this time, additional medium was added to the packaging cells to generate more virus. After 7 hours, the second pool of viral supernatant was prepared as the first, except with 4 μg/ml polybreen. U-937 cells were resuspended in this viral supernatant and the infection was allowed to proceed overnight. Then the U-937 cells were placed into fresh medium without any selection. After 18 hours, the medium was changed to selection medium containing 0.4 μg/ml puromycin. The pool of stably transfected clones was propagated for binding and chemotaxis assays.

Equilibrium binding assays

Equilibrium binding assay was conducted in RPMI 1640 medium with 10 mM HEPES and 0.2% BSA in room temperature with constant shaking for 2 hours. Reaction mixture contained a total volume of 100 μl with various concentrations of ¹²⁵I-labeled ligands (¹²⁵I-human MCP-1 or ¹²⁵I-mouse JE), U-937/rabbit CCR2 stable transfectants (10⁵ cells /well for ¹²⁵I-human MCP-1 and 10⁴ cells /well for ¹²⁵I-mouse JE) in the presence or absence of corresponding cold ligand. Nonspecific binding was determined by 100-fold excess of cold ligand. Reactions were stopped by separation using the glass fiber filter (Wallac Printed Filtermat A) on a Tomtec Harvester 96-2 (Hamden, CT). Filters were subsequently washed 5 times with wash buffer (10 mM HEPES, 500 mM NaCl, pH 7.4) to remove the unbound radioligand. Finally, bound radioligand was quantitated using a liquid scintillation counter (Wallac 1205 Betaplate, Perkin Elmer). Dissociation constant (K _d ) and maximum binding (B _max ) were calculated by non-linear regression with Graphpad Prism 4.01 (San Diego, CA).

Competition binding assays

The assay was conducted under the identical conditions as saturation binding assay described above with the radiolabeled ligand concentration fixed (0.11 nM ¹²⁵I-mouse JE) in the presence of various concentrations of unlabeled chemokines or TAK-779. Values of IC₅₀ were calculated by fitting the competition curves with Graphpad Prism 4.01.

Chemotaxis assays

Chemotaxis assay was performed as described elsewhere [18] with a few modifications. Parental U-937 or U-937/rabbit CCR2 stable transfectants were harvested, counted and resuspended at a final cell density of 5 × 10⁶ /ml in chemotaxis buffer (RPMI 1640 medium, containing 0.3% BSA and 1 mM HEPES). Chemokines were diluted to 1000 ng/ml stock and serially diluted (1:3) in migration buffer in a Costar 96 well plate (Cat #3790, Costar, Corning NY). Thirty microliters of each chemokine dilution were transferred to the corresponding well in the bottom chamber of a ChemoTX 96 well migration plate (5 μM pore size; Neuroprobe, Gaithersburg, MD). The ChemoTX filter unit was assembled and 50 μl of cells were added to the top chamber and incubated for 2.5 hours. Plates were then removed and cells were aspirated off the filter top. The cells that had migrated to the bottom chamber were pulsed with 5 μl of Aqueous One Solution (Promega, Madison, WI) and incubated for an additional 30–60 minutes at 37°C. Plates were shaken gently to permit uniform mixing and read at 490 nm in a Spectra_max Plus reader (Molecular Dynamics, Piscataway, NJ). Cell standard curves were incorporated into the bottom of each plate with cell number ranges from 0–150,000 cells/well. The results were expressed as percent cell migration versus chemokine concentration. For compound testing, TAK-779 was diluted in DMSO at various concentrations and added to both the top and bottom chambers and chemotaxis assay was performed in the presence of 4 nM of mouse JE as described above.