Cutting Edge: Expression of the C-C Chemokine Receptor CCR3 in Human Airway Epithelial Cells This information is current as Cristiana Stellato, Mary E. Brummet, James R. Plitt, Syed of September 28, 2021. Shahabuddin, Fuad M. Baroody, Mark C. Liu, Paul D. Ponath and Lisa A. Beck J Immunol 2001; 166:1457-1461; ; doi: 10.4049/jimmunol.166.3.1457 http://www.jimmunol.org/content/166/3/1457 Downloaded from References This article cites 29 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/166/3/1457.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 28, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. ● Cutting Edge: Expression of the C-C Chemokine Receptor CCR3 in Human Airway Epithelial Cells1 Cristiana Stellato,* Mary E. Brummet,* James R. Plitt,* Syed Shahabuddin,* Fuad M. Baroody,† Mark C. Liu,* Paul D. Ponath,‡ and Lisa A. Beck2* an anti-CCR3 Ab blocked Ͼ95% of the eosinophil response to Chemokine-induced eosinophil chemotaxis is mediated primarily CCR3 agonists in vitro (6). In animal studies, CCR3 blockade Downloaded from through the C-C chemokine receptor, CCR3. We have now de- significantly inhibited eotaxin- and Ag-induced eosinophil accu- tected CCR3 immunoreactivity on epithelial cells in biopsies of mulation (7). CCR3 mRNA and protein levels have been found to patients with asthma and other respiratory diseases. CCR3 be significantly elevated in the bronchial mucosa and skin of pa- mRNA was detected by Northern blot analysis after TNF-␣ stim- tients with asthma (8) and atopic dermatitis (9), respectively. ulation of the human primary bronchial epithelial cells as well as Chemokine receptors have been demonstrated on structural ␥ ␣ the epithelial cell line, BEAS-2B; IFN- potentiated the TNF- - cells, such as smooth muscle and endothelial cells (10, 11). We http://www.jimmunol.org/ induced expression. Western blots and flow cytometry confirmed now report that human airway epithelial cells express a functional the expression of CCR3 protein. This receptor is functional based CCR3. Epithelial cells play a significant role in the chemokine .(on studies demonstrating eotaxin-induced intracellular Ca2؉ flux network, as a major source of both CXC and C-C chemokines (12 and tyrosine phosphorylation of cellular proteins. The specificity Among the C-C chemokines, epithelial cells produce the CCR3 of this functional response was confirmed by blocking these sig- agonists, RANTES, MCP-3, MCP-4, eotaxin, and eotaxin-2 (13– naling events with anti-CCR3 mAb (7B11) or pertussis toxin. 16). These C-C chemokines may contribute to the accumulation Furthermore, 125I-eotaxin binding assay confirmed that CCR3 and activation of eosinophils and other inflammatory cells in the expressed on epithelial cells have the expected ligand specificity. allergic airway. The coexpression of CCR3 and its ligands suggest These studies indicate that airway epithelial cells express CCR3 that epithelial cells may have a C-C chemokine autoregulatory by guest on September 28, 2021 and suggest that CCR3 ligands may influence epithelial cell pathway. functions. The Journal of Immunology, 2001, 166: 1457–1461. Materials and Methods Immunohistochemical tissue staining he C-C chemokine receptor CCR3 plays a critical role in Formalin-fixed, paraffin-embedded tissue sections were stained using Vec- allergic inflammation. It is highly expressed on eosino- tastain ABC-AP Kit and Red Substrate Kit (Vector Laboratories, Burlin- phils (1), basophils (2), microglial cells (3), and mono- game, CA). The mouse monoclonal blocking anti-CCR3 Ab (7B11; Leu- T koSite, Boston, MA) anti-eotaxin (2G6; LeukoSite), and anti-RANTES cyte-derived dendritic cells (4), and its expression has also been (3D3; Genentech, San Francisco, CA) were used with the isotype-matched reported in a subset of Th2 lymphocytes (5). CCR3 mediates the (IgG2a; Coulter-Immunotech, Miami, FL) mouse Ig control. potent chemotactic and activating effects of eotaxin, eotaxin-2, eotaxin-3, RANTES, monocyte chemoattractant protein (MCP)3-3, Cell culture and MCP-4 on eosinophils. Treatment of human eosinophils with The BEAS-2B and the 16-HBE cell lines were kindly donated by Drs. Curtis Harris and Dieter Gruenert, respectively (17, 18). Primary bronchial epithelial cells (PBEC) were isolated and purity confirmed as described *Division of Clinical Immunology and Allergy, Johns Hopkins Asthma and Allergy (19, 20). Normal human bronchial epithelial cells (NHBE) were used as Center, Baltimore, MD 21224; †Division of Otolaryngology, University of Chicago, another source of primary epithelial cells (CC-2541; Clontech, Palo Alto, Chicago, IL; and ‡LeukoSite Inc., Boston, MA 02142 CA). BEAS-2B (passage 33–40) and PBEC (passage 1) were cultured in Received for publication December 13, 1999. Accepted for publication December Hanks’ F12/DMEM medium with 5% heat-inactivated FCS, 1% L-glu- 8, 2000. tamine, 1% fungizone, penicillin (100 U/ml), and streptomycin (100 mg/ The costs of publication of this article were defrayed in part by the payment of page ml). 16-HBE (passage 18–22) were cultured in DMEM with 10% heat- charges. This article must therefore be hereby marked advertisement in accordance inactivated FCS, 1% L-glutamine, 1% fungizone, penicillin (100 U/ml), with 18 U.S.C. Section 1734 solely to indicate this fact. and streptomycin (100 mg/ml), and NHBE (passage 1–3) were cultured in BEGM Bullet Kit (Clontech). 1 This work was supported by National Institutes of Health Grants AI 01226 and AI 44242-01. Northern blot analysis 2 Address correspondence and reprint request to Dr. Lisa A. Beck, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224- Total RNA was extracted with RNAzol B (20). RNA (20 ␮g) was elec- 6801. E-mail address: [email protected] trophoresed and blotted onto Genescreen plus nylon membranes (NEN Life 32 3 Abbreviations used in this paper: MCP, monocyte chemoattractant protein; ERK, Sciences, Boston, MA). Membranes were hybridized with a P-labeled extracellular signal-regulated kinase; NHBE, normal human bronchial epithelial cells; cDNA probe encoding 0.3 kb of the CCR3 coding region, or a GAPDH PBEC, primary bronchial epithelial cells; MIP, macrophage inflammatory protein; probe (Clontech), and washed with high-stringency conditions (2ϫ SSC, MFI, mean fluorescence intensity. 0.1% SDS, 65°C, 15 min). Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 ● 1458 CUTTING EDGE Western blot analysis for CCR3 protein trol Ig, washed, and then incubated with saturating dilutions of FITC-conju- gated F(abЈ) goat anti-mouse IgG Ab (Tago, Burlingame, CA). Cell lysates (10 ␮g/lane) were separated by SDS-PAGE and transferred to 2 a Sequi-blot polyvinylidene difluoride membrane (Bio-Rad, Hercules, Cytosolic Ca2ϩ measurements CA). Blots were blocked in 1ϫ PBS/5% BSA/0.1% Tween 20 overnight and incubated with the anti-CCR3 Ab 7B11 (1 ␮g/ml) or with the rabbit Cells were loaded with 4 mM fura-2AM (Molecular Probes, Eugene, OR) polyclonal anti-CCR3 Ab H-52 (1 ␮g/ml; Santa Cruz Biotechnology, Santa for 60 min at 37°C in Hanks’ F12/DMEM. Cells were then washed, incu- Cruz, CA), washed with 1ϫ PBS/0.1% Tween 20, and incubated with bated with HBSS buffer, and viewed under a Zeiss digital video micro- HRP-conjugated secondary Ab. Immunoreactive bands were visualized us- scope (Oberkochen, Germany) before and after stimulation with 10 nM ␮ ing ECL (Amersham, Arlington Heights, IL). eotaxin and a positive control, bradykinin (1 M). 125 Stimulation, preparation of cytosolic extracts, and Western I chemokine binding assay analysis Confluent BEAS-2B grown in 24-well plates were washed with binding buffer (25 mM HEPES, 8 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.5% Epithelial cells were washed with serum-free media and preincubated with BSA, 0.1% sodium azide, pH 7.8) before incubation (90 min, room tem- ␮ ␮ either anti-CCR3 Ab (7B11, 10 g/ml) or IgG2a (10 g/ml; Coulter) for perature) with 1–1.5 ϫ 105 cpm of 125I-eotaxin (Amersham) with increas- ␮ 30 min on ice or pertussis toxin (1 g/ml; Sigma, St. Louis, MO) for 1 h ing concentrations of unlabeled eotaxin (R&D Systems) or 100 nM of at 37°C. Cells were stimulated with eotaxin (R&D Systems, Minneapolis, either macrophage inflammatory protein (MIP)-1␣ (BioSource, Camarillo, MN) for up to 10 min. The reaction was quenched with cold 10 mM CA) or IL-8 (PeproTech, Rocky Hill, NJ). Cells were washed in buffer (25 sodium orthovanadate (Sigma) and complete mini-protease inhibitor cock- mM HEPES, 1 mM MgCl2, 1 mM CaCl2, 0.5 M NaCl, 0.1% sodium azide, tail tablets (Boehringer Mannheim, Indianapolis, IN). The cells were har- pH 7.8) and lysed in buffer with 1% Triton X-100. Free and bound ligands vested and lysed with lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 5 were separated using the Brandel cell harvester (Bethesda, MD) and What- mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM sodium orthovanadate, man GF/F filters (Tewksbury, MA), blocked with polyethylenimine (0.2% Downloaded from and protease inhibitor tablets).