High expression of the chemokine receptor CCR3 in human blood basophils. Role in activation by eotaxin, MCP-4, and other chemokines. M Uguccioni, … , M Baggiolini, C A Dahinden J Clin Invest. 1997;100(5):1137-1143. https://doi.org/10.1172/JCI119624. Research Article Eosinophil leukocytes express high numbers of the chemokine receptor CCR3 which binds eotaxin, monocyte chemotactic protein (MCP)-4, and some other CC chemokines. In this paper we show that CCR3 is also highly expressed on human blood basophils, as indicated by Northern blotting and flow cytometry, and mediates mainly chemotaxis. Eotaxin and MCP-4 elicited basophil migration in vitro with similar efficacy as regulated upon activation normal T cells expressed and secreted (RANTES) and MCP-3. They also induced the release of histamine and leukotrienes in IL-3- primed basophils, but their efficacy was lower than that of MCP-1 and MCP-3, which were the most potent stimuli of exocytosis. Pretreatment of the basophils with a CCR3-blocking antibody abrogated the migration induced by eotaxin, RANTES, and by low to optimal concentrations of MCP-4, but decreased only minimally the response to MCP-3. The CCR3-blocking antibody also affected exocytosis: it abrogated histamine and leukotriene release induced by eotaxin, and partially inhibited the response to RANTES and MCP-4. In contrast, the antibody did not affect the responses induced by MCP-1, MCP-3, and macrophage inflammatory protein-1alpha, which may depend on CCR1 and CCR2, two additional receptors detected by Northern blotting with basophil RNA. This study demonstrates that CCR3 is the major receptor for eotaxin, RANTES, and MCP-4 in human basophils, and suggests that basophils and eosinophils, which are the characteristic […] Find the latest version: https://jci.me/119624/pdf High Expression of the Chemokine Receptor CCR3 in Human Blood Basophils Role in Activation by Eotaxin, MCP-4, and Other Chemokines Mariagrazia Uguccioni,* Charles R. Mackay,‡ Brigitte Ochensberger,§ Pius Loetscher,ʈ Silvia Rhis,§ Gregory J. LaRosa,‡ Patricia Rao,‡ Paul D. Ponath,‡ Marco Baggiolini,* and Clemens A. Dahinden§ *Theodor Kocher Institute, University of Bern, CH-3000 Bern 9, Switzerland; ‡LeukoSite, Inc., Cambridge, Massachusetts 02142; and §Institute of Immunology and Allergology, ʈDivision of Rheumatology, University Hospital, CH-3010 Bern, Switzerland Abstract generally activate monocytes, lymphocytes, eosinophils, and basophils (2). All chemokines act via seven transmembrane Eosinophil leukocytes express high numbers of the chemo- domain, G protein–coupled receptors (3), and five of them, kine receptor CCR3 which binds eotaxin, monocyte chemo- CCR1–CCR5, are known to mediate responses to CC chemo- tactic protein (MCP)-4, and some other CC chemokines. In kines. The best-characterized ligands are regulated upon acti- this paper we show that CCR3 is also highly expressed on vation normal T cells expressed and secreted (RANTES),1 human blood basophils, as indicated by Northern blotting macrophage inflammatory protein (MIP)-1␣, and monocyte and flow cytometry, and mediates mainly chemotaxis. Eo- chemotactic protein (MCP)-3 for CCR1 (4–7), MCP-1 and taxin and MCP-4 elicited basophil migration in vitro with other MCPs for CCR2 (6, 8, 9), eotaxin, RANTES, and MCP-3 similar efficacy as regulated upon activation normal T cells for CCR3 (10–12), MIP-1␣ and RANTES for CCR4 (13), and expressed and secreted (RANTES) and MCP-3. They also in- MIP-1␣, MIP-1␤, and RANTES for CCR5 (14–16). The ex- duced the release of histamine and leukotrienes in IL-3–primed pression of CC chemokine receptors differs depending on the basophils, but their efficacy was lower than that of MCP-1 type of leukocyte, and influences responsiveness. It is the basis and MCP-3, which were the most potent stimuli of exocyto- for the selectivity observed in the migration of circulating leu- sis. Pretreatment of the basophils with a CCR3-blocking an- kocytes into inflamed tissues in response to chemokines (3). tibody abrogated the migration induced by eotaxin, RANTES, Since eosinophils and basophils are major effector cells in and by low to optimal concentrations of MCP-4, but de- allergic inflammation (17), we have studied two recently char- creased only minimally the response to MCP-3. The CCR3- acterized, powerful eosinophil attractants, eotaxin and MCP-4, blocking antibody also affected exocytosis: it abrogated his- as potential agonists for basophils. Eotaxin has been reported tamine and leukotriene release induced by eotaxin, and to be selective for eosinophils (18) and to act via CCR3 (19). partially inhibited the response to RANTES and MCP-4. In MCP-4 has similar properties, but also acts on monocytes and contrast, the antibody did not affect the responses induced lymphocytes, like other MCPs and RANTES (17, 20–26). In by MCP-1, MCP-3, and macrophage inflammatory protein- this paper, we show that eotaxin and MCP-4 are powerful 1␣, which may depend on CCR1 and CCR2, two additional stimuli for basophils and induce chemotaxis and the release of receptors detected by Northern blotting with basophil RNA. histamine and peptido-leukotrienes. These activities are medi- This study demonstrates that CCR3 is the major receptor ated mainly via CCR3, which is highly expressed both in eosin- for eotaxin, RANTES, and MCP-4 in human basophils, and ophils and basophils. suggests that basophils and eosinophils, which are the char- acteristic effector cells of allergic inflammation, depend Methods largely on CCR3 for migration towards different chemo- kines into inflamed tissues. (J. Clin. Invest. 1997. 100:1137– Chemokines. All chemokines were prepared by automated solid- 1143.) Key words: allergy • inflammation • chemotaxis phase chemical synthesis on a peptide synthesizer (430A; Applied Biosystems, Inc., Foster City, CA) using the tertiary butyloxycarbonyl Introduction and benzyl protection strategy (27). Recombinant human IL-3 was kindly provided by Sandoz, Ltd. (Basel, Switzerland). Chemokines are divided into two main subfamilies according Monoclonal antibodies. The mAb of IgG2a isotype 7B11, which blocks CCR3, was generated as previously described (19). An mAb to the position of the first two cysteines which are either sepa- against the ␣ chain of the high-affinity IgE-receptor (anti-Fc⑀RI) was rated by one amino acid or adjacent (CXC and CC chemo- generously provided by Dr. Hakimi and Dr. Chizzonite (Hoffman-La kines) (1). CXC chemokines attract mainly neutrophils and Roche, Nutley, NJ) (28). FITC-labeled anti-CD9 was purchased from lymphocytes, whereas CC chemokines are more versatile and Serotec Ltd. (Kidlington, Oxford, United Kingdom). Cells. Basophils from venous blood of healthy volunteers (29) were enriched (10–45%) by fractionation in a discontinuous Percoll Address correspondence to Dr. Clemens A. Dahinden, Institute of gradient, and further purified by negative selection with magnetic Immunology and Allergology, University Hospital, Inselspital, CH- beads coated with mAbs against CD3, CD4, CD8, CD14, CD16, and 3010 Bern, Switzerland. Phone: 41-31-632-8244; FAX: 41-31-381- CD19 (30). The final cell suspension consisted of 70–80% basophils 5735; E-mail: [email protected] and 20–30% lymphocytes. Basophils used for RNA extraction were Received for publication 13 March 1997 and accepted in revised purified to near homogeneity by an additional positive selection step form 20 May 1997. using FITC-labeled anti-Fc⑀RI followed by magnetic beads coated J. Clin. Invest. © The American Society for Clinical Investigation, Inc. 1. Abbreviations used in this paper: LTC4, leukotriene C4; MCP, 0021-9738/97/09/1137/07 $2.00 monocyte chemotactic protein; MIP, macrophage inflammatory pro- Volume 100, Number 5, September 1997, 1137–1143 tein; RANTES, regulated upon activation normal T cells expressed http://www.jci.org and secreted. Basophil Activation via CCR3 1137 with anti-FITC. Eosinophils (Ͼ 98% pure) and IL-2–expanded T binding studies, the CCR3 transfectants were treated overnight with 2 lymphocytes were obtained as described previously (25, 31). mM sodium butyrate before harvesting by centrifugation. The cells (5 ϫ Flow cytometry. Mixtures of basophils and mononuclear cells 105) were incubated at room temperature for 90 min with 0.1 nM and of neutrophils and eosinophils were obtained by Percoll gradient 125I-labeled eotaxin or MCP-1 in 100 ␮l binding buffer (50 mM centrifugation without negative selection. The cells suspended in PBS Hepes, pH 7.2, 1 mM CaCl2, 5 mM MgCl2, 0.5% BSA, and 0.02% so- supplemented with 10% FCS and 1 mM EDTA were incubated with dium azide), in the presence of increasing concentrations of unla- anti-CCR3 or isotype-matched control mAb for 30 min on ice and beled chemokines. Bound and free tracer were separated by filtration then treated with PE-conjugated goat anti–mouse Fab (Southern using 96-well GF/B Unifilter plates (Packard, Meriden, CT) pre- Biotechnology Associates, Inc., Birmingham, AL). Two-color stain- soaked in 0.3% polyethylenimine. The filters were washed at 4ЊC ing was performed with FITC-labeled anti-Fc⑀RI for basophils, and with 300 ␮l binding buffer supplemented with 0.5 M NaCl to reduce with FITC-labeled anti-CD9 for eosinophils. After fixation, the cells nonspecific binding. All assays were done in duplicate. IC50 values were analyzed by flow cytometry (FACScan®, Becton Dickinson, (concentrations at which binding of the iodinated chemokine was in- Mountain View, CA). For estimation of anti-CCR3 binding sites, 100 hibited by 50%) were calculated by a three-parameter logistic curve fit ␮l heparinized blood diluted with 100 ␮l PBS containing 0.1% azide using KaleidaGraph software (Synergy Software, Inc., Reading, PA). was incubated at room temperature with 400 ng FITC-labeled anti- CCR3 and 500 ng biotin-coupled anti–human IgE (PharMingen, San Diego, CA). After washing, the cells were stained by addition of 5 ␮l Results Streptavidin-Quantum red (Sigma Chemical Co., St. Louis, MO). Histamine and leukotriene release. In former studies, we have Erythrocytes were lysed, and the leukocytes analyzed by flow cytom- etry.