Functional Expression and Characterization of Macaque C-C Chemokine Receptor 3 (CCR3) and Generation of Potent Antagonistic Anti-macaque CCR3 Monoclonal Antibodies
2002; Elsevier BV; Volume: 277; Issue: 37 Linguagem: Inglês
10.1074/jbc.m205488200
ISSN1083-351X
AutoresLiwen Zhang, Marco P. Soares, Yanfen Guan, Stephen Matheravidathu, Richard Wnek, Kristine Johnson, Anna Meisher, Susan A. Iliff, John S. Mudgett, Martin S. Springer, Bruce L. Daugherty,
Tópico(s)Antimicrobial Peptides and Activities
ResumoEosinophils are major effector cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The β-chemokine receptor C-C chemokine receptor 3 (CCR3) provides a mechanism for the selective recruitment of eosinophils into tissue and thus has recently become an attractive biological target for therapeutic intervention. In order to develop in vivo models of inflammatory diseases, it is essential to identify and characterize the homologues of human eotaxin (C-C chemokine ligand 11) and CCR3 from other species, such as non-human primates. Accordingly, we cloned the macaque eotaxin and CCR3 genes and revealed that they were 91 and 92% identical at the amino acid level to their human homologues, respectively. Macaque CCR3 expressed in the murine pre-B L1-2 cell line bound macaque eotaxin with high affinity (Kd = 0.1 nm) and exhibited a robust eotaxin-induced Ca2+ flux and chemotaxis. Characterization of β-chemokines on native macaque CCR3 on eosinophils was performed by means of eotaxin-induced shape change in whole blood using a novel signaling assay known as gated autofluorescence forward scatter. Additionally, mAbs were raised against macaque CCR3 using two different immunogens: a 30-amino acid synthetic peptide derived from the predicted NH2 terminus of macaque CCR3 and intact macaque CCR3-transfected cells. These anti-macaque CCR3 monoclonal antibodies exhibited potent antagonist activity in receptor binding and functional assays. The characterization of the macaque eotaxin/CCR3 axis and development of antagonistic anti-macaque CCR3 monoclonal antibodies will facilitate the development of CCR3 small molecule antagonists with the hope of ameliorating chronic inflammatory diseases in humans. Eosinophils are major effector cells implicated in a number of chronic inflammatory diseases in humans, particularly bronchial asthma and allergic rhinitis. The β-chemokine receptor C-C chemokine receptor 3 (CCR3) provides a mechanism for the selective recruitment of eosinophils into tissue and thus has recently become an attractive biological target for therapeutic intervention. In order to develop in vivo models of inflammatory diseases, it is essential to identify and characterize the homologues of human eotaxin (C-C chemokine ligand 11) and CCR3 from other species, such as non-human primates. Accordingly, we cloned the macaque eotaxin and CCR3 genes and revealed that they were 91 and 92% identical at the amino acid level to their human homologues, respectively. Macaque CCR3 expressed in the murine pre-B L1-2 cell line bound macaque eotaxin with high affinity (Kd = 0.1 nm) and exhibited a robust eotaxin-induced Ca2+ flux and chemotaxis. Characterization of β-chemokines on native macaque CCR3 on eosinophils was performed by means of eotaxin-induced shape change in whole blood using a novel signaling assay known as gated autofluorescence forward scatter. Additionally, mAbs were raised against macaque CCR3 using two different immunogens: a 30-amino acid synthetic peptide derived from the predicted NH2 terminus of macaque CCR3 and intact macaque CCR3-transfected cells. These anti-macaque CCR3 monoclonal antibodies exhibited potent antagonist activity in receptor binding and functional assays. The characterization of the macaque eotaxin/CCR3 axis and development of antagonistic anti-macaque CCR3 monoclonal antibodies will facilitate the development of CCR3 small molecule antagonists with the hope of ameliorating chronic inflammatory diseases in humans. bronchoalveolar lavage C-C chemokine receptor C-X-C chemokine receptor regulated upon activation, normal T-cell expressed and secreted monocyte chemotactic protein gated autofluorescence forward scatter monoclonal antibody airway hyperreactivity peripheral blood mononuclear cell phosphate-buffered saline Bronchial asthma is a multifactorial disease characterized clinically by reversible bronchoconstriction leading to shortness of breath. In the pathophysiology of the disease, a chronic inflammatory condition persists in the airways of most patients, which involves a complex interplay between blood leukocytes, airway epithelial cells, and bronchial smooth muscle cells. One of the most striking aspects of asthma is the selective accumulation and activation of distinct subtypes of leukocytes into the airways, particularly eosinophils, and it is these cells that are postulated to play a key role in the pathophysiology of the disease (1Gleich G.J. Adolphson C.R. Leiferman K.M. Gallin J.I. Goldstein I.M. Snyderman R. Inflammation: Basic Principles and Clinical Correlates. 2nd Ed. Raven Press, Ltd., New York1992: 663-700Google Scholar, 2Seminario M.C. Gleich G.J. Curr. Opin. Immunol. 1994; 6: 860-864Crossref PubMed Scopus (130) Google Scholar). Inflammatory mediators, such as chemoattractants, generated at the involved sites, promote the migration of eosinophils from the vasculature into the tissue. Unlike eicosanoids and complement cleavage fragments, which display activities on a wide variety of cells, candidate molecules for the selective recruitment of eosinophils into the airways are a class of proteins called chemotactic cytokines or chemokines. Chemokines are a growing superfamily of >50 small molecular mass proteins (∼8–10 kDa) and are characterized by their actions on distinct subtypes of leukocytes (3Mackay C.R. Nat. Immunol. 2001; 2: 95-101Crossref PubMed Scopus (718) Google Scholar, 4Luster A.D. N. Engl. J. Med. 1998; 338: 436-445Crossref PubMed Scopus (3272) Google Scholar). These proteins can be classified into two major subfamilies based on the arrangement of the first two conserved cysteines in the protein. In the α or C-X-C family, these two cysteines are separated by any amino acid, whereas in the β or C-C family, these two cysteines are adjacent to one another. Some members of this latter family have been discovered to possess strong eosinophil migratory and activating properties. Chemokines exert their effects by binding to members of the G-protein-coupled receptor superfamily of receptors, which contain seven transmembrane domains. 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Moreover, we have generated murine monoclonal antibodies directed against the macaque CCR3 using two different immunizing antigens and have demonstrated that these mAbs are potent functional antagonists of this receptor through a series of receptor binding and signaling assays in vitro. Genomic DNA was isolated from venous blood of cynomolgus 2All animal protocols were approved by the Institutional Animal Care and Use Committee and were conducted in accordance with applicable local and federal animal welfare regulations. and rhesus macaques (Macaca fascicularis and Macaca mulatta, respectively; Merck Research Laboratories, in-house colony) using the QIAamp DNA blood kit (Qiagen, Valencia, CA). Also, cynomolgus macaque DNA was purchased from Therion (Troy, NY), and rhesus macaque DNA was purchased from CLONTECH (Palo Alto, CA). Southern blot hybridization was performed using standard procedures (41Maniatis T. Fritsch E.F. Sambrook J. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, 1982: 382-389Google Scholar). Briefly, genomic DNA (20 μg) was digested with a set of restriction enzymes for 7 h, followed by phenol/chloroform extraction, ethanol precipitation, and then separation on a 0.7% agarose/Tris acetate-EDTA gel. The gel was then saturated in 1.5 m NaCl and 0.5 m NaOH (to denature the DNA) for 45 min followed by neutralization in 1.5m NaCl and 1 m Tris-Cl (pH 8.0) for 45 min. DNA was transferred onto a Hybond-N+ membrane (AmershamBiosciences) using 20× SSC as transfer buffer. Prehybridization was carried out at 42 °C for 1 h in 6× SSC, 5× Denhardt’s solution, 0.5% SDS, 50% formamide, and 100 μg/ml salmon sperm DNA, followed by hybridization for 16 h in the identical solution containing 2 × 106 cpm/ml 32P-labeled 1.1-kb human CCR3 DNA fragment (Ready-to-Go DNA labeling kit; AmershamBiosciences) comprising the open reading frame (7Daugherty B.L. Siciliano S.J. DeMartino J.A. Malkowitz L. Sirotina A. Springer M.S. J. Exp. Med. 1996; 183: 2349-2354Crossref PubMed Scopus (502) Google Scholar). The membrane was washed twice in 2× SSC, 0.1% SDS at room temperature for 15 min; twice in 0.1× SSC, 0.1% SDS at room temperature for 15 min; once at 55 °C for 15 min; and once at 65 °C for 15 min. The membrane was then exposed onto Eastman Kodak Co. X-OMAT film and developed. PCR was performed using cynomolgus and rhesus macaque genomic DNA as template with primers designed from the 5′- and 3′-untranslated regions of the human CCR3 gene. The 5′-primer contained the sequence −23 to −3 bp upstream of the ATG initiation codon and a HindIII site (5′-GGC-TTA-AGC-TTC-TAT-CAC-AGG-GAG-AAG-TG-3′). The 3′-primer contained the sequence 11–29 bp downstream of the TAG termination codon and aNotI site (5′-CTT-CAT-CTC-CTT-GCG-GCC-GCT-CCT-CTT-TAG-GCA-ATT-TTC-3′). PCR was performed for 30 cycles: 94 °C for 60 s, 55 °C for 60 s, and 72 °C for 2 min in a PerkinElmer Life Sciences model 9600 DNA thermal cycler. The resultant PCR products from the cynomolgus and rhesus DNA were subcloned into expression vector pBJ-Neo (7Daugherty B.L. Siciliano S.J. DeMartino J.A. Malkowitz L. Sirotina A. Springer M.S. J. Exp. Med. 1996; 183: 2349-2354Crossref PubMed Scopus (502) Google Scholar) and pcDEF3 (42Goldman L.A. Cutrone E.C. Kotenko S.V. Krause C.D. Langer J.A. BioTechniques. 1996; 21: 1013-1015Crossref PubMed Scopus (152) Google Scholar) (a generous gift from Dr. J. Langer, University of Medicine and Dentistry of New Jersey), respectively. Poly(A)+mRNA was isolated by oligo(dT)-cellulose chromatography from rhesus macaque small intestine and used to generate first strand cDNA using a tagged oligo(dT) primer (3′-RACE kit; Invitrogen). The cDNA was then used as template for PCR performed with the following two primers: 5′-primer (5′-AAC-CAC-CAC-CTC-TCA-CGC-C-3′) and 3′-primer (5′-CAG-GCT-CTG-GTT-TGG-TTT-CAA-3′). PCR was performed for 30 cycles: 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min. The resultant PCR product was subcloned into pCR2.1-TOPO (Invitrogen). A consensus sequence for rhesus macaque eotaxin was identified by DNA sequencing of six independent clones. The rhesus macaque eotaxin protein was chemically synthesized (Gryphon Sciences, South San Francisco, CA) based on the predicted amino acid sequence. The murine pre-B L1-2 cell line was a generous gift from Dr. I. Weissman (Stanford University). Transfection of the L1-2 and AML14.3D10 cell lines were carried out as described (7Daugherty B.L. Siciliano S.J. DeMartino J.A. Malkowitz L. Sirotina A. Springer M.S. J. Exp. Med. 1996; 183: 2349-2354Crossref PubMed Scopus (502) Google Scholar, 8Ponath P.D. Qin S. Post T.W. Wang J., Wu, L. Gerard N.P. Newman W. Gerard C. Mackay C.R. J. Exp. Med. 1996; 183: 2437-2448Crossref PubMed Scopus (553) Google Scholar) with slight modifications. Briefly, 5 × 106 cells were washed in Hanks’ balanced saline solution, mixed with 20 μg of pcDEF3-rhesus CCR3 or pcDEF3-human CCR3 (for L1-2 transfection), and pBJ/Neo-cynomolgus CCR3 (for AML14.3D10 transfection) in a 0.4-cm electroporation cuvette, electroporated at 250 V and 960 microfarads, and then cultured in complete medium (RPMI 1640 with 10% fetal bovine serum). After 48 h, the cells were placed in medium containing 0.8 mg/ml G418 and plated onto 96-well plates at 18,000 cells/well. Clones were transferred into six-well plates, and positive clones were selected by their ability to bind125I-human eotaxin (Amersham Biosciences). 125I-Human eotaxin (2000 Ci/mmol) was obtained from PerkinElmer Life Sciences, and125I-macaque eotaxin (2000 Ci/mmol) was custom-radioiodinated at Amersham Biosciences. L1-2/macaque CCR3 cells (30,000 cells/assay for 125I-macaque eotaxin and 200,000 cells/assay for 125I-human eotaxin) were mixed with 30 pm (∼20,000 cpm) iodinated chemokine and varying concentrations of β-chemokines (PeproTech, Rocky Hill, NJ) in a binding buffer consisting of 50 mm Hepes, pH 7.2, 1 mm CaCl2, 5 mm MgCl2, 0.1% gelatin and incubated at 30 °C for 60 min essentially as described (7Daugherty B.L. Siciliano S.J. DeMartino J.A. Malkowitz L. Sirotina A. Springer M.S. J. Exp. Med. 1996; 183: 2349-2354Crossref PubMed Scopus (502) Google Scholar). Radioactivity retained on polyethyleneimine-treated Whatman GFC membranes after washing in binding buffer (using a PerkinElmer Life Sciences harvester) containing 0.5m NaCl was counted in a TopCount (PerkinElmer Life Sciences). Binding results were analyzed using KaleidaGraph (Synergy Software, Reading, PA) and LIGAND (43Munson P.J. Rodbard D. Anal. Biochem. 1980; 107: 220-239Crossref PubMed Scopus (7772) Google Scholar). Changes in intracellular calcium level were measured on a fluorescence imaging plate reader (FLIPR; excitation 488-nm argon laser line/emission 530-nm bandpass interference filter; Molecular Devices, Inc., Sunnyvale, CA) following the manufacturer’s instructions with modifications. L1-2/macaque CCR3 or L1-2/human CCR3 cells were washed in wash buffer (Hanks’ balanced saline solution containing 20 mm Hepes, pH 7.2, 0.1% bovine serum albumin) and labeled with fluo-3 (Molecular Probes, Inc., Eugene, OR) at 1.5 × 106 cells/ml in labeling buffer at 37 °C for 1 h. Labeling buffer was prepared as follows. 50 μg of fluo-3 was dissolved in 44 μl of 10% pluronic F-127 in Me2SO and then added to 11 ml of wash buffer. Following labeling with fluo-3, cells were washed twice in wash buffer. About 1.5 × 105 cells in 135 μl of wash buffer were added to each well of a 96-well plate (black, clear bottom; Corning Costar, Cambridge, MA) and then centrifuged for 5 min (no brake). The plate was placed on FLIPR, 67.5-μl ligands (one-half of the cell volume) at various concentrations were added, and fluorescence changes were recorded. For blocking experiments, mAbs were preincubated with cells for 20 min before 3 nm macaque eotaxin was added. Results were calculated using KaleidaGraph. L1-2/macaque CCR3 cells were labeled with 5 μm calcein (Molecular Probes), washed, and resuspended in chemotaxis buffer (RPMI 1640 plus 0.5% bovine serum albumin) at 5 × 106 cells/ml. To the bottom chamber of a 96-well Neuroprobe ChemoTx plate (5-μm pore size; NeuroProbe, Inc., Gaithersburg, MD), increasing concentrations of chemokine were added in a volume of 29 μl of chemotaxis buffer. To the top chamber, 30 μl of cells (1.5 × 105 total cells) were added, and chemotaxis was allowed to proceed at 37 °C for 1 h. Unmigrated cells were removed from membrane with a Kimwipe. The plate was then analyzed in a CytoFluor II fluorometer (excitation, 485 nm; emission, 530 nm; PerSeptive Biosystems, Framingham, MA). For blocking experiments, 5 nm macaque eotaxin was added to the bottom chamber. To the top chamber, 15 μl of cells (1.5 × 105 total) was mixed together with 15 μl of mAb at various concentrations. GAFS assays were performed as described (44Sabroe I. Hartnell A. Jopling L.A. Bel S. Ponath P.D. Pease J.E. Collins P.D. Williams T.J. J. Immunol. 1999; 162: 2946-2955Crossref PubMed Google Scholar) 3T. Hansel, personal communication. with modifications for whole blood. Briefly, 90 μl of fresh rhesus macaque blood was mixed with varying concentrations of chemokines (in 10 μl) in 1.2-ml polypropylene tubes, incubated at 37 °C for 10 min, and then transferred onto ice. To preserve the shape change, the cells were fixed in 250 μl of ice-cold optimized fixative solution containing 2.5% Cytofix (BD Biosciences, San Jose, CA), 22.5% H2O, and 75% FACSflow (BD Biosciences) and left on ice for 2 min. The mixture was then transferred into 1 ml of ice-cold lysis buffer (155 mm NH4Cl, 10 mm KHCO3) in a Falcon tube (catalog no. 2052) and left on ice for another 20 min to achieve uniform red cell lysis. For blocking experiments, aliquots of blood were preincubated with mAbs in a total volume of 90 μl for 20 min at room temperature before 10 μl of macaque eotaxin (7.5 nm final concentration) was added. The samples were run on a FACScan flow cytometer (BD Biosciences), and data from 30,000 cells from each sample were collected. Eosinophils were gated out based on their high autofluorescence, and mean forward scatter was calculated by Consort 40/VAX software (Becton Dickinson, Immunocytometry Systems). Typically, more than 4,500 eosinophils in each sample were analyzed. The mean forward scatter of the eosinophils in each sample was determined, and a dose-response curve for each chemokine or mAb was generated accordingly. Two immunizing antigens were used to generate monoclonal antibodies against the macaque CCR3. The first approach used viable whole L1-2 cells stably expressing the full-length macaque CCR3 sequence as immunogen, essentially as described (27Heath H. Qin S. Rao P., Wu, L. LaRosa G. Kassam N. Ponath P.D. Mackay C.R. J. Clin. Invest. 1997; 99: 178-184Crossref PubMed Scopus (440) Google Scholar) with some modifications. About 1.5 × 107 L1-2/rhesus macaque CCR3 cells were injected into C57b/6 mice. Intraperitoneal injections were performed four times at 2-week intervals (Cell Essentials, Boston, MA). Postimmune mouse sera were tested by flow cytometry and inhibition of125I-human eotaxin binding on AML14.3D10-cynomolgus macaque CCR3 transfectants versus untransfected AML14.3D10 cells. The most potent serum blocked 125I-human eotaxin binding by 50% at 1:1000 serum dilution and blocked this binding completely at 1:125 dilution (data not shown). The mice whose sera were most potent in flow cytometry and inhibition of 125I-human eotaxin binding were chosen for fusion. The final injection of whole L1-2/rhesus macaque CCR3 cells was performed intravenously. Three days postinjection, mouse splenocytes were fused with SP2/0 myeloma cells. Approximately 1600 hybridoma clones were obtained from two fusions (Cell Essentials). To eliminate any clones that reacted to native L1-2 cell surface antigens, screening of hybridoma supernatants was performed by flow cytometry on AML14.3D10/cynomolgus macaque CCR3 cells. In the second approach, a peptide (30-mer) was synthesized (Gryphon Sciences), corresponding to the predicted NH2terminus of the macaque CCR3 amino acid sequence (TTS-LDT-VET-FGP-TSY-DDD-MGL-LCE-KAD-VGA-norleucinal-amide). The peptide was conjugated to thyroglobulin and injected into BALB/c mice at 3-week intervals. Mouse sera were tested by flow cytometry on L1-2/macaque CCR3 cells versus L1-2 untransfected cells and inhibition of 125I-human eotaxin binding to L1-2/macaque CCR3 cells. Postimmune sera possessed similar potency as those using whole L1-2/macaque CCR3 cells as immunogen. As above, the mouse that produced the most potent serum was used for fusion. A total of about 600 hybridoma clones were obtained by this method from a single fusion. Positive hybridoma clones were selected by enzyme-linked immunosorbent assay (see below) and tested by flow cytometry on L1-2/macaque CCR3 cells. Positive clones identified from both approaches tested positive on L1-2/macaque CCR3 and negative on the parental L1-2 cell line by flow cytometry. Hybridoma clones producing anti-macaque CCR3 mAbs were subcloned to ensure purity and then injected into mice for ascites production. The IgG fraction from the ascites fluid was purified by protein A chromatography and dialyzed against PBS. Antibody concentration was determined by the Bradford protein assay kit (Bio-Rad). The isotype of the anti-macaque CCR3 mAbs was determined by enzyme-linked immunosorbent assay. About 5 × 105 cells (L1.2/rhesus macaque CCR3, AML14.3D10/cynomolgus macaque CCR3, or partially purified rhesus eosinophils or PBMCs) were washed in staining buffer (PBS, 5% fetal bovine serum); subsequently, normal goat IgG (10 μg/ml final concentration, ICN Pharmaceuticals, Costa Mesa, CA) was added and incubated at room temperature for 10 min. Hybridoma supernatant (50 μl), purified anti-macaque CCR3 mAbs, mouse IgG (ICN) (negative control), or mouse anti-human CD11b (BD Biosciences; clone ICRF44), all at 3 μg/ml, was then added to the cells incubated at room temperature for 30 min. Cells were washed, and then fluorescein isothiocyanate-labeled goat F(ab′)2 anti-mouse IgG (5 μg/ml; ICN) was added to the cells and incubated at room temperature for 30 min. For the rhesus macaque PBMCs, R-phycoerythrin-conjugated mouse anti-human CD3 mAb (clone SP34; BD Biosciences) was incubated as above. The cells were washed and resuspended at 106/ml in staining buffer containing 1 μg/ml propidium iodide. Fluorescein isothiocyanate fluorescence was analyzed on a FACScan flow cytometer or FACSCalibur (BD Biosciences). Bovine serum albumin-conjugated macaque CCR3 NH2-terminal peptide (4 μg/ml in PBS) was loaded onto 96-well plates (100 μl/well) and incubated at 4 °C for 18 h. The plates were then washed three times with PBS. Blocking solution (3% fish gel in PBS, 300 μl/well) was added, and the plates were incubated at room temperature for 2 h. The plates were then washed, and hybridoma supernatant was added to each well and incubated for 1 h. The supernatant was removed, and the plates were washed in PBS containing 0.05% (v/v) Nonidet P-40. Horseradish peroxidase enzyme-conjugated secondary antibody was added to each well, and the plates were incubated for 30 min. The plates were washed again in PBS containing 0.05% Nonidet P-40. ABTS-100 substrate solution was then added and incubated for 30 min for color development. Clones that gave a signal/noise ratio of greater than 3 were picked for further analysis. Rhesu
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