title: Human eosinophils express functional CCR7.
abstract: Human eosinophils display directed chemotactic activity toward an array of soluble chemokines. Eosinophils have been observed to migrate to draining lymph nodes in experimental models of allergic inflammation, yet it is unknown whether eosinophils express CCR7, a key chemokine receptor in coordinating leukocyte trafficking to lymph nodes. The purpose of this study is to demonstrate expression of CCR7 by human eosinophils and functional responses to CCL19 and CCL21, the known ligands of CCR7. Human eosinophils were purified by negative selection from healthy donors. CCR7 expression of freshly purified, unstimulated eosinophils and of IL-5-primed eosinophils was determined by flow cytometry and Western blot. Chemotaxis to CCL19 and CCL21 was measured in transwell assays. Shape changes to CCL19 and CCL21 were analyzed by flow cytometry and microscopy. Calcium fluxes of fluo-4 AM-loaded eosinophils were recorded by flow cytometry after chemokine stimulation. ERK phosphorylation of CCL19- and CCL21-stimulated eosinophils was measured by Western blot and Luminex assay. Human eosinophils expressed CCR7 as demonstrated by flow cytometry and Western blots. Eosinophils exhibited detectable cell surface expression of CCR7. IL-5-primed eosinophils exhibited chemotaxis toward CCL19 and CCL21 in a dose-dependent fashion. Upon stimulation with CCL19 or CCL21, IL-5-primed eosinophils demonstrated dose-dependent shape changes with polarization of F-actin and exhibited calcium influxes. Finally, primed eosinophils stimulated with CCL19 or CCL21 exhibited increased phosphorylation of ERK in response to both CCR7 ligands. We demonstrate that human eosinophils express CCR7 and have multipotent responses to the known ligands of CCR7.
authors: Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA. pakuthot@bidmc.harvard.edu
authors: Praveen
authors: P
authors: Akuthota
authors: Shigeharu
authors: S
authors: Ueki
authors: Jessica
authors: J
authors: Estanislau
authors: Peter F
authors: PF
authors: Weller
mesh_terms: D000199:Actins / Q000378:metabolism; D000814:Aniline Compounds / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D048430:Cell Shape; D054415:Chemokine CCL19 / Q000276:immunology / Q000494:pharmacology; D054421:Chemokine CCL21 / Q000276:immunology / Q000494:pharmacology; D002633:Chemotaxis; D004305:Dose-Response Relationship, Drug; D004804:Eosinophils / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D015848:Interleukin-5 / Q000378:metabolism; D010766:Phosphorylation; D054400:Receptors, CCR7 / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014966:Xanthenes / Q000378:metabolism
chemical_list: D000199:Actins; D000814:Aniline Compounds; C515460:CCL19 protein, human; C515478:CCL21 protein, human; C515370:CCR7 protein, human; D054415:Chemokine CCL19; D054421:Chemokine CCL21; C409648:Fluo 4; C508599:IL5 protein, human; D015848:Interleukin-5; D054400:Receptors, CCR7; D011994:Recombinant Proteins; D014966:Xanthenes; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium