Airway Epithelial Cells Chemokine Receptor CCR3 in Human Cutting

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

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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).

● 1458 CUTTING EDGE

Western blot analysis for CCR3 protein trol Ig, washed, and then incubated with saturating dilutions of FITC-conjugated 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). Protein concentrations were determined us- solution, 1 h). ing the bicinchoninic acid assay (Pierce, Rockford, IL). Samples were boiled in 4ϫ SDS buffer (0.5 M Tris, pH 6.8, 16% glycerol, 3% SDS, 8% Results 2-ME, 2 mg bromophenol blue) and 10 ␮g of protein loaded onto a 10% Tris-glycine SDS polyacrylamide gel. Proteins were transferred to a poly- We performed immunohistochemical analysis of inflammatory and vinylidene difluoride membrane and washed in PBST (20 mM Tris, 137 noninflammatory biopsies from human tissues using an Ab di- mM NaCl, 0.2% Tween 20), and nonspecific binding was blocked with 5% rected against CCR3, 7B11. As expected, we observed eosinophil BSA (Fisher Scientific, Pittsburgh, PA) in PBST. The membranes were staining (Fig. 1A). Surprisingly, intense staining of the airway ep- http://www.jimmunol.org/ incubated overnight (4°C) with p42/p44 anti-phospho-extracellular signal- ϭ related kinase (ERK) or p42/p44 anti-ERK (New England Biolabs, Bev- ithelium was observed in biopsies of asthmatic subjects (n 6; erly, MA) or anti-phosphotyrosine Ab, clone 4G10 (Upstate Biotechnol- Fig. 1C). This epithelial CCR3 immunoreactivity is not at odds ogy, Lake Placid, NY), all at 1/1000 dilution. Equal loading of cell lysates with other published studies (8, 22, 23) but can be explained by was reconfirmed by both amido black (Bio-Rad) and p42/p44 anti-ERK differences in tissue fixation. We found that epithelial CCR3 stain- staining. Membranes were probed as noted in Western blot methods. ing is entirely lost when tissues are fixed in 4% paraformaldehyde Flow cytometry and snap-frozen, whereas the same tissues stain intensely for CCR3 when fixed in 10% buffered formalin and paraffin-embed- Epithelial cells were labeled by indirect immunofluorescence and analyzed ded (not shown). Staining for the CCR3 ligands, eotaxin and RAN- using the EPICS Profile II flow cytometer (Coulter Electronics, Hialeah, FL) by guest on September 28, 2021 as described (21). Cells were incubated in saturating concentrations of the TES, on serial sections correlated significantly with epithelial anti-CCR3 mAb 7B11, or an equivalent concentration of isotype-matched con- CCR3 immunoreactivity. This was best illustrated in an open lung

FIGURE 1. CCR3 immunoreactivity in eosinophils and airway epithelium. Photomicrographs (ϫ400) of CCR3 staining of (A) perivascular eosinophils in the dermis of an allergic subject 30 min after cutaneous RANTES challenge; (C) the epithelium and endothelium from a bronchial biopsy of an unchallenged asthmatic subject (representative of n ϭ 5). The immunoreactivity of CCR3 (E) and two of its ligands, eotaxin (F) and RANTES (G), were closely correlated in an open lung biopsy from a patient with idiopathic hypereosinophilic syndrome. No staining was observed with control Abs (B, D, and H). Micrographs E–H represent serial sections from the same tissue block (ϫ200). The Journal of Immunology 1459 biopsy from a patient with idiopathic hypereosinophilic syndrome from allergic donors (MFI fold control ϭ 11.6 Ϯ 1.4, n ϭ 15; not (Fig. 1, E–H). Twelve of 16 nasal polyp samples showed epithelial shown). immunoreactivity for CCR3. The atopic status of these subjects In two of five experiments, a sharp increase in intracellular Ca2ϩ (eight atopic and eight nonatopic) did not predict the extent or was observed in unstimulated BEAS-2B cells loaded with fura-2 intensity of CCR3 staining. and challenged with the CCR3 agonist, eotaxin (10 nM) and was To analyze epithelial CCR3 expression in vitro, we stimulated similar to that seen with bradykinin, which also binds to a seven- ϭ BEAS-2B (Fig. 2A) and PBEC (n 2, not shown) for 24 h with transmembrane G protein-coupled receptor (Fig. 3A). Eotaxin ␣ TNF- (100 ng/ml) and found induction of CCR3 mRNA by stimulation also induced tyrosine phosphorylation of cellular pro- ␥ Northern blot analysis. Treatment of cells with IFN- for 24 h did teins including members of the mitogen-activated protein kinase not induce CCR3 mRNA; however, IFN-␥ potentiated TNF-␣- family, which was maximal at 0.5 min (Fig. 3B; n ϭ 3 primary induced CCR3 mRNA expression, which increased under such cells and n ϭ 3 in 16-HBE (not shown)). These signaling events stimulation in a concentration-dependent fashion (Fig. 2B). In two were inhibited by either pretreatment with 7B11 or pertussis toxin experiments, incubation with IL-4 (50 ng/ml) or IL-10 (10 ng/ml) (n ϭ 3 primary cells and n ϭ 3 in 16-HBE (not shown)). for 24 h did not induce CCR3 mRNA. IL-4 partially inhibited ␣ In competition binding assays, cold eotaxin potently inhibited TNF- -induced CCR3 expression up to 39%, while IL-10 mod- 125 estly up-regulated TNF-␣-induced CCR3 up to 30% ( p ϭ NS, not the binding of I-eotaxin binding to BEAS-2B cells (Fig. 4). The ␣ shown). CCR3 mRNA was weakly detected in unstimulated C-C chemokine MIP-1 , which does not bind to CCR3, and the BEAS-2B cells in one of three experiments. In contrast with CXC chemokine IL-8 did not displace radiolabeled eotaxin bind- mRNA expression, the levels of CCR3 protein detected by West- ing even at high concentrations (100 nM). Downloaded from ern blot analysis of resting and TNF-␣ plus IFN-␥-stimulated BEAS-2B cells (Fig. 2C) or 16-HBE cells (not shown) were very similar. Similarly, CCR3 surface expression was detected by flow cytometry on unstimulated BEAS-2B cells (mean fluorescence intensity (MFI) fold control ϭ 1.55 Ϯ 0.13, n ϭ 11) and was slightly

increased by stimulation with TNF-␣ plus IFN-␥ (MFI fold con- http://www.jimmunol.org/ trol ϭ 1.69 Ϯ 0.19, n ϭ 10). Unstimulated 16-HBE cells (MFI fold control ϭ 1.70 Ϯ 0.13, n ϭ 12) and PBECs (MFI fold control ϭ 1.52 Ϯ 0.16, n ϭ 3) expressed similar levels of CCR3 (Fig. 2D). Epithelial CCR3 levels were considerably less than on eosinophils by guest on September 28, 2021

FIGURE 2. Detection of CCR3 in BEAS-2B cells. A, Northern blot analysis of BEAS-2B cells treated with control medium, TNF-␣ (100 ng/ ml) or IFN-␥ (100 ng/ml) alone or in combination for 24 h. I, Autoradiog- raphy of CCR3 mRNA expression (1.6 kb; representative of n ϭ 5); II, control GAPDH mRNA expression. B, Densitometric analysis of Northern FIGURE 3. CCR3 functional assays. A, A sharp intracellular Ca2ϩ flux blot assay showing increasing expression of CCR3 mRNA in BEAS-2B was noted in BEAS-2B cells in response to eotaxin and bradykinin (inset; cells upon stimulation with increasing concentrations of TNF-␣ plus IFN-␥ positive control). B, Western blot analysis demonstrating the kinetics of -p Ͻ 0.05 compared with unstimulated cells). C, Western blot phosphorylation of the p42 mitogen-activated protein kinase in eotaxin ,ء ;n ϭ 4) analysis (representative of n ϭ 3), performed using the same Ab used for stimulated PBECs. PBECs were preincubated with media alone or pertussis immunohistochemistry (7B11). Identical results were obtained when using toxin (1 ␮g/ml, 1 h) and stimulated with eotaxin (100 ng/ml) for up to 1 the polyclonal Ab, H-52 (n ϭ 2, data not shown). Purified (Ͼ98% purity) min. Similar results were noted when lysates were analyzed by immunoblot human eosinophils (Eos) or neutrophils (Neuts) were used as positive and for eotaxin-induced tyrosine phosphorylation with and without preincuba- negative controls, respectively. D, Surface expression of CCR3 on PBEC tion with the CCR3-blocking mAb 7B11. (These are representative blots of (top; representative of n ϭ 3) and BEAS-2B (bottom; representative of n ϭ n ϭ 3 for primary epithelial cells and n ϭ 3 for 16-HBE cells.) To ensure 11). Histograms of flow cytometric analysis with anti-CCR3 (7B11) are equal loading in each lane, we load each lane with 10 ␮g of total protein represented by the dark line histograms and the IgG2a isotype control are (measured by the bicinchoninic acid assay) and run a parallel immunoblot depicted by the light line histograms. probed with p42/p44 anti-ERK Ab (data not shown). 1460 CUTTING EDGE

diate epithelial cell migration and proliferation, which would be important in tissue remodeling. Chemokine receptors have been subverted for use as entry molecules by numerous intracellular pathogens such as malaria (27), HIV (28), and poxvirus (29); thus, epithelial CCR3 may play a role in microbial infections.

Acknowledgments We acknowledge the important advice obtained from Dr. Bruce S. Bochner for the ﬂow cytometric studies, Dr. Daniel J. Dairaghi and Dr. David Proud for the radiolabeled ligand binding studies, Dr. Donald W. MacGlashan for intracy- toplasmic calcium ﬂux experiments, Dr. Narashimha Parinandi for intracellular signaling studies, and Dr. Robert P. Schleimer and Dr. Peter Jeffery for helpful discussions. We also thank Lynette Sholl and Stephanie L. Curry for their skillful assistance in cell culturing and Ca2ϩ signaling studies.

References 125 FIGURE 4. Competitive inhibition of I-eotaxin binding to BEAS-2B 1. Combadiere, C., S. K. Ahuja, and P. M. Murphy. 1995. Cloning and functional cells. BEAS-2B cells (representative of n ϭ 2) were incubated with 0.3 nM expression of a human eosinophil CC chemokine receptor. J. Biol. Chem. 270: of 125I-eotaxin in the absence (E) or presence of 50 pM to 1 mM of un- 16491. Downloaded from labeled eotaxin (F) or 100 nM IL-8 (Ⅺ) or MIP-1␣ (f). After 90 min at 2. Uguccioni, M., C. R. Mackay, B. Ochensberger, P. Loetscher, S. Rhis, G. J. LaRosa, P. Rao, P. D. Ponath, M. Baggiolini, and C. A. Dahinden. 1997. 4°C, cells were washed and radioactivity counted as described in Materials High expression of the chemokine receptor CCR3 in human blood basophils. and Methods. J. Clin. Invest. 100:1137. 3. He, J., Y. Chen, M. Farzan, H. Choe, A. Ohagen, S. Gartner, J. Busciglio, Z. Yang, W. Hogmann, W. Newman, et al. 1997. CCR3 and CCR5 are coreceptors for HIV-1 infection of microglia. Nature 385:645. 4. Rubbert, A., C. Combadiere, M. Ostrowski, J. Arthos, M. Dybul, E. Machado,

Discussion M. A. Cohn, J. A. Hoxie, P. M. Murphy, and A. S. Fauci. 1998. Dendritic cells http://www.jimmunol.org/ Chemokines and their receptors mediate the selective recruitment express multiple chemokine receptors used as coreceptors for HIV entry. J. Im- munol. 160:3933. of leukocytes into inflammatory tissues. For example, the eosino- 5. Sallusto, F., C. R. Mackay, and A. Lanzavecchia. 1997. Selective expression of phil-rich infiltrate seen in allergic diseases is due in part to the the eotaxin receptor CCR3 by human T helper 2 cells. Science 277:2005. effects of CCR3 agonists on eosinophils. In this paper, we report 6. Heath, H., S. Qin, P. Rao, L. Wu, G. LaRosa, N. Kassam, P. D. Ponath, and C. R. Mackay. 1997. Chemokine receptor usage by human eosinophils. J. Clin. that 1) human airway epithelial cells express cell surface CCR3, 2) Invest. 99:178. this expression can be modulated by inflammatory cytokines, and 7. Sabroe, I., D. M. Conroy, N. P. Gerard, Y. Li, P. D. Collins, T. W. Post, P. J. Jose, T. J. Williams, C. J. Gerard, and P. D. Ponath. 1998. Cloning and characterization 3) epithelial CCR3 transduces intracellular signals. of the guinea pig eosinophil eotaxin receptor, C-C chemokine receptor-3: block- We noted concordant expression in the airway epithelium of ade using a monoclonal antibody in vivo. J. Immunol. 161:6139. CCR3 with two of its ligands, eotaxin and RANTES. This suggests 8. Ying, S., D. S. Robinson, Q. Meng, J. Rottman, R. Kennedy, D. J. Ringler, by guest on September 28, 2021 C. R. Mackay, B. L. Daugherty, M. S. Springer, S. R. Durham, T. J. Williams, that in vivo, CCR3 is dynamically regulated and that the expres- and A. B. Kay. 1997. Enhanced expression of eotaxin and CCR3 mRNA and sion of CCR3 and its ligands share some common regulatory ele- protein in atopic asthma: association with airway hyperresponsiveness and pre- ments. 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