Mechanisms of Allergy
Receptor internalization is required for eotaxin-induced responses in human eosinophils,☆☆

https://doi.org/10.1067/mai.2003.3Get rights and content

Abstract

Background: CC chemokine receptor 3 (CCR3) is a major chemokine receptor involved in regulating eosinophil trafficking, and therefore the elucidation of ligand-induced CCR3 events has important implications in understanding the biologic and pathologic properties of eosinophils. After ligand binding to CCR3, cellular signals include stimulatory (ie, calcium mobilization, actin polymerization, shape change, and chemotaxis) and inhibitory (ie, desensitization of the receptor) events. We have previously demonstrated that CCR3 undergoes rapid and prolonged ligand-induced internalization. Objective: Here we explore the role of internalization in downstream cellular processes, including shape change, actin polymerization, calcium mobilization, and desensitization. Methods: Peripheral blood–derived human eosinophils were pretreated with 2 mechanistically distinct inhibitors of internalization, sucrose and phenylarsine oxide, and functional responses were monitored. Results: We first demonstrate that ligand-induced internalization is required for chemokine-induced eosinophil shape change. To define which signaling components upstream of eosinophil shape change required internalization, we next studied the role of internalization in calcium mobilization and actin polymerization. Sucrose and phenylarsine oxide pretreatment inhibited actin polymerization, implicating receptor internalization in this early response. In contrast, calcium mobilization was not inhibited by blockade of internalization. Finally, we were interested in testing the role of internalization in receptor desensitization. We first demonstrated that preincubation with eotaxin induced a dose-dependent desensitization in eotaxin-induced eosinophil transepithelial migration. However, this phenomenon was not inhibited by blockade of internalization. Conclusion: These results establish that CCR3 internalization is critically involved in select eosinophil functional responses (ie, cellular shape change and actin polymerization) but not desensitization and calcium mobilization. (J Allergy Clin Immunol 2003;111:97-105.)

Section snippets

Eosinophil purification

Eosinophils were purified from healthy or mildly atopic volunteers by means of negative immunomagnetic selection on the basis of the method of Hansel et al.20 Briefly, granulocytes were isolated from heparin-anticoagulated whole blood by means of dextran sedimentation, Percoll centrifugation, and hypotonic lysis of red blood cells. Cells were resuspended in HBSS (Gibco BRL) with 2% FCS and incubated with 0.75 μL/106 cells anti-CD16–conjugated microbeads (MACS, Miltenyi Biotech Inc) for 30

Role of internalization in eotaxin-induced eosinophil shape change

We have previously shown that CCR3 undergoes rapid ligand-induced internalization.13, 21 The functional importance of this receptor-trafficking event is largely unknown. We hypothesized that internalization is required for downstream functional responses after CCR3 stimulation. Initially, we examined the role of internalization in chemokine-induced eosinophil shape change.24 In this assay granulocytes were exposed to eotaxin for a brief period of time, and the change in shape of eosinophils

Discussion

CCR3 is rapidly internalized into the early endosome compartment after agonist stimulation in a manner similar to that observed for several other GPCRs13, 36; however, the functional significance of this event has not been previously elucidated. In this article we report several novel findings concerning the function of internalization in eosinophils. First, we report that ligand-induced CCR3 internalization is inhibited by PAO and sucrose. These 2 distinct well-established inhibitors of

Acknowledgements

We thank Patricia Fulkerson, Laura Kindinger, and Jessica Kavanaugh for excellent technical assistance. We also thank Drs Gurjit Hershey, David Williams, and Fred Finkelman for critical discussions and Andrea Lippelman for editorial assistance.

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    Supported in part by the American Heart Association Scientist Development Grant (N.Z.), National Institutes of Health grants R01 AI42242-05 (M.E.R.) and R01 AI45898-03 (M.E.R.), the Human Frontier Science Program (M.E.R), International Life Sciences Institute (M.E.R.), and Burroughs Wellcome Fund (M.E.R.). NZ is a Parker B. Francis fellow.

    ☆☆

    Reprint requests: Marc E. Rothenberg, MD, PhD, Division of Allergy and Immunology, Children's Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH 45229.

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