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 pMSCVpuro 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 pMSCVpuro, 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 Spectramax 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.
