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+ cells (25).
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85069408829
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note
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-7 M phorbol 12-myristate 13-acetate (PMA). Cytokine production was measured in the 24-hour culture supernatant by enzyme-linked immunosorbent assay (ELISA), using matched pairs of antibodies specific for IL-2, IL-4, IL-5, IL-10, tumor necrosis factor-α (TNF-α), TNF-β, and IFN-γ (PharMingen, San Diego, CA).
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-7 M PMA plus ionomycin (1 μg/ml) for 4 hours. Brefeldin A (10 μg/ml) was added during the last 2 hours. Cells were fixed with 2% paraformaldehyde, permeabilized with phosphate-buffered saline containing fetal bovine serum (1%) and saponin (0.5%) and stained with fluorescein isothiocyanate (FITC)-labeled anti-IFN-γ (IgG1) and phycoerythrin (PE)-labeled anti-IL-4 (IgG2b) monoclonal antibodies (Becton Dickinson, Mountain View, CA). In some experiments, the cells were stained, before permeabilization, with anti-CD4 (BL4, IgG2a, Immunotech, Marseille, France) or anti-CD8 (OKT8, IgG2a, American Type Culture Collection), then with biotin-labeled goat anti-mouse IgG2a (Southern Biotechnology, Birmingham, AL) and streptavidin-tricolor (Molecular Probes, Eugene, OR). Because T cell activation results in a rapid down-regulation of CCR3, it is not possible to assess CCR3 expression and intracellular cytokines simultaneously.
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note
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+ cells. Results comparable to those in Fig. 1 were obtained in sorted cell lines obtained from two healthy and three atopic individuals. In three out of five cases the CCR3-depleted cells produced lower concentrations of IL-4 and IL-5 than the unsorted cells. No clear differences were found in the production of IL-10, IFN-γ, IL-2, TNF-α, or TNF-β, or between healthy and atopic donors. A correlation between CCR3 expression and production of IL-4 but not IFN-γ was observed in 13 antigen-specific T cell clones (14).
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unpublished data
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F. Sallusto, unpublished data.
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unpublished data
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C. R. MacKay, unpublished data. Chemotaxis experiments were done as described by P. D. Ponath et al. (7), with a transendothelial assay and eotaxin or MCP-1 at 10 nM and IP-10 at 20 nM. Cells migrating to the bottom of the transwell chemotaxis chamber were counted by flow cytometry.
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MacKay, C.R.1
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Eotaxin is not the only agonist for CCR3, because MCP-3, MCP-4, RANTES (2), and eotaxin 2 [U. Forrsmann et al., J. Exp. Med. 185, 2171 (1997)] can also bind and trigger CCR3.
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note
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We thank A. Mari and F. Nestle for providing samples from allergic patients; M. Dessing and A. Pickert for cell sorting; D. Lenig and H. Heath for expert technical assistance; and M. Cella, M. Colonna, and M. Kopf for critical reading and comments. The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche Ltd., Basel, Switzerland. F. S. is also with the department of immunology, Istituto Superiore de Sanità, Rome.
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