Mechanisms of AllergyReceptor internalization is required for eotaxin-induced responses in human eosinophils☆,☆☆
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.
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Reprint requests: Marc E. Rothenberg, MD, PhD, Division of Allergy and Immunology, Children's Hospital Medical Center, 3333 Burnet Ave, Cincinnati, OH 45229.