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The Journal of Immunology, 2001, 166: 1457-1461.
Copyright © 2001 by The American Association of Immunologists

CUTTING EDGE

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{dagger}, Mark C. Liu*, Paul D. Ponath{ddagger} and Lisa A. Beck2,*

* Division of Clinical Immunology and Allergy, Johns Hopkins Asthma and Allergy Center, Baltimore, MD 21224; {dagger} Division of Otolaryngology, University of Chicago, Chicago, IL; and {ddagger} LeukoSite Inc., Boston, MA 02142


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chemokine-induced eosinophil chemotaxis is mediated primarily through the C-C chemokine receptor, CCR3. We have now detected CCR3 immunoreactivity on epithelial cells in biopsies of patients with asthma and other respiratory diseases. CCR3 mRNA was detected by Northern blot analysis after TNF-{alpha} stimulation of the human primary bronchial epithelial cells as well as the epithelial cell line, BEAS-2B; IFN-{gamma} potentiated the TNF-{alpha}-induced expression. Western blots and flow cytometry confirmed the expression of CCR3 protein. This receptor is functional based on studies demonstrating eotaxin-induced intracellular Ca2+ flux and tyrosine phosphorylation of cellular proteins. The specificity of this functional response was confirmed by blocking these signaling events with anti-CCR3 mAb (7B11) or pertussis toxin. Furthermore, 125I-eotaxin binding assay confirmed that CCR3 expressed on epithelial cells have the expected ligand specificity. These studies indicate that airway epithelial cells express CCR3 and suggest that CCR3 ligands may influence epithelial cell functions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The C-C chemokine receptor CCR3 plays a critical role in allergic inflammation. It is highly expressed on eosinophils (1), basophils (2), microglial cells (3), and monocyte-derived dendritic cells (4), and its expression has also been reported in a subset of Th2 lymphocytes (5). CCR3 mediates the potent chemotactic and activating effects of eotaxin, eotaxin-2, eotaxin-3, RANTES, monocyte chemoattractant protein (MCP)3-3,and MCP-4 on eosinophils. Treatment of human eosinophils with an anti-CCR3 Ab blocked >95% of the eosinophil response to CCR3 agonists in vitro (6). In animal studies, CCR3 blockade significantly inhibited eotaxin- and Ag-induced eosinophil accumulation (7). CCR3 mRNA and protein levels have been found to be significantly elevated in the bronchial mucosa and skin of patients with asthma (8) and atopic dermatitis (9), respectively.

Chemokine receptors have been demonstrated on structural cells, such as smooth muscle and endothelial cells (10, 11). We now report that human airway epithelial cells express a functional CCR3. Epithelial cells play a significant role in the chemokine network, as a major source of both CXC and C-C chemokines (12). Among the C-C chemokines, epithelial cells produce the CCR3 agonists, RANTES, MCP-3, MCP-4, eotaxin, and eotaxin-2 (13, 14, 15, 16). These C-C chemokines may contribute to the accumulation and activation of eosinophils and other inflammatory cells in the allergic airway. The coexpression of CCR3 and its ligands suggest that epithelial cells may have a C-C chemokine autoregulatory pathway.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Immunohistochemical tissue staining

Formalin-fixed, paraffin-embedded tissue sections were stained using Vectastain ABC-AP Kit and Red Substrate Kit (Vector Laboratories, Burlingame, CA). The mouse monoclonal blocking anti-CCR3 Ab (7B11; LeukoSite, Boston, MA) anti-eotaxin (2G6; LeukoSite), and anti-RANTES (3D3; Genentech, San Francisco, CA) were used with the isotype-matched (IgG2a; Coulter-Immunotech, Miami, FL) mouse Ig control.

Cell culture

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 (19, 20). Normal human bronchial epithelial cells (NHBE) were used as another source of primary epithelial cells (CC-2541; Clontech, Palo Alto, CA). BEAS-2B (passage 33–40) and PBEC (passage 1) were cultured in Hanks’ F12/DMEM medium with 5% heat-inactivated FCS, 1% L-glutamine, 1% fungizone, penicillin (100 U/ml), and streptomycin (100 mg/ml). 16-HBE (passage 18–22) were cultured in DMEM with 10% heat-inactivated FCS, 1% L-glutamine, 1% fungizone, penicillin (100 U/ml), and streptomycin (100 mg/ml), and NHBE (passage 1–3) were cultured in BEGM Bullet Kit (Clontech).

Northern blot analysis

Total RNA was extracted with RNAzol B (20). RNA (20 µg) was electrophoresed and blotted onto Genescreen plus nylon membranes (NEN Life Sciences, Boston, MA). Membranes were hybridized with a 32P-labeled cDNA probe encoding 0.3 kb of the CCR3 coding region, or a GAPDH probe (Clontech), and washed with high-stringency conditions (2x SSC, 0.1% SDS, 65°C, 15 min).

Western blot analysis for CCR3 protein

Cell lysates (10 µg/lane) were separated by SDS-PAGE and transferred to a Sequi-blot polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA). Blots were blocked in 1x PBS/5% BSA/0.1% Tween 20 overnight and incubated with the anti-CCR3 Ab 7B11 (1 µg/ml) or with the rabbit polyclonal anti-CCR3 Ab H-52 (1 µg/ml; Santa Cruz Biotechnology, Santa Cruz, CA), washed with 1x PBS/0.1% Tween 20, and incubated with HRP-conjugated secondary Ab. Immunoreactive bands were visualized using ECL (Amersham, Arlington Heights, IL).

Stimulation, preparation of cytosolic extracts, and Western analysis

Epithelial cells were washed with serum-free media and preincubated with either anti-CCR3 Ab (7B11, 10 µg/ml) or IgG2a (10 µg/ml; Coulter) for 30 min on ice or pertussis toxin (1 µg/ml; Sigma, St. Louis, MO) for 1 h at 37°C. Cells were stimulated with eotaxin (R&D Systems, Minneapolis, MN) for up to 10 min. The reaction was quenched with cold 10 mM sodium orthovanadate (Sigma) and complete mini-protease inhibitor cocktail tablets (Boehringer Mannheim, Indianapolis, IN). The cells were harvested and lysed with lysis buffer (10 mM Tris, pH 7.4, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 1% Triton X-100, 1 mM sodium orthovanadate, and protease inhibitor tablets). Protein concentrations were determined using the bicinchoninic acid assay (Pierce, Rockford, IL). Samples were boiled in 4x SDS buffer (0.5 M Tris, pH 6.8, 16% glycerol, 3% SDS, 8% 2-ME, 2 mg bromophenol blue) and 10 µg of protein loaded onto a 10% Tris-glycine SDS polyacrylamide gel. Proteins were transferred to a polyvinylidene difluoride membrane and washed in PBST (20 mM Tris, 137 mM NaCl, 0.2% Tween 20), and nonspecific binding was blocked with 5% BSA (Fisher Scientific, Pittsburgh, PA) in PBST. The membranes were incubated overnight (4°C) with p42/p44 anti-phospho-extracellular signal-related kinase (ERK) or p42/p44 anti-ERK (New England Biolabs, Beverly, MA) or anti-phosphotyrosine Ab, clone 4G10 (Upstate Biotechnology, Lake Placid, NY), all at 1/1000 dilution. Equal loading of cell lysates was reconfirmed by both amido black (Bio-Rad) and p42/p44 anti-ERK staining. Membranes were probed as noted in Western blot methods.

Flow cytometry

Epithelial cells were labeled by indirect immunofluorescence and analyzed using the EPICS Profile II flow cytometer (Coulter Electronics, Hialeah, FL) as described (21). Cells were incubated in saturating concentrations of the anti-CCR3 mAb 7B11, or an equivalent concentration of isotype-matched control Ig, washed, and then incubated with saturating dilutions of FITC-conjugated F(ab')2 goat anti-mouse IgG Ab (Tago, Burlingame, CA).

Cytosolic Ca2+ measurements

Cells were loaded with 4 mM fura-2AM (Molecular Probes, Eugene, OR) for 60 min at 37°C in Hanks’ F12/DMEM. Cells were then washed, incubated with HBSS buffer, and viewed under a Zeiss digital video microscope (Oberkochen, Germany) before and after stimulation with 10 nM eotaxin and a positive control, bradykinin (1 µM).

125I chemokine binding assay

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% BSA, 0.1% sodium azide, pH 7.8) before incubation (90 min, room temperature) with 1–1.5 x 105 cpm of 125I-eotaxin (Amersham) with increasing concentrations of unlabeled eotaxin (R&D Systems) or 100 nM of either macrophage inflammatory protein (MIP)-1{alpha} (BioSource, Camarillo, CA) or IL-8 (PeproTech, Rocky Hill, NJ). Cells were washed in buffer (25 mM HEPES, 1 mM MgCl2, 1 mM CaCl2, 0.5 M NaCl, 0.1% sodium azide, pH 7.8) and lysed in buffer with 1% Triton X-100. Free and bound ligands were separated using the Brandel cell harvester (Bethesda, MD) and Whatman GF/F filters (Tewksbury, MA), blocked with polyethylenimine (0.2% solution, 1 h).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We performed immunohistochemical analysis of inflammatory and noninflammatory biopsies from human tissues using an Ab directed against CCR3, 7B11. As expected, we observed eosinophil staining (Fig. 1GoA). Surprisingly, intense staining of the airway epithelium was observed in biopsies of asthmatic subjects (n = 6; Fig. 1GoC). This epithelial CCR3 immunoreactivity is not at odds with other published studies (8, 22, 23) but can be explained by differences in tissue fixation. We found that epithelial CCR3 staining is entirely lost when tissues are fixed in 4% paraformaldehyde and snap-frozen, whereas the same tissues stain intensely for CCR3 when fixed in 10% buffered formalin and paraffin-embedded (not shown). Staining for the CCR3 ligands, eotaxin and RANTES, on serial sections correlated significantly with epithelial CCR3 immunoreactivity. This was best illustrated in an open lung biopsy from a patient with idiopathic hypereosinophilic syndrome (Fig. 1Go, E–H). Twelve of 16 nasal polyp samples showed epithelial immunoreactivity for CCR3. The atopic status of these subjects (eight atopic and eight nonatopic) did not predict the extent or intensity of CCR3 staining.



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  		FIGURE 1. CCR3 immunoreactivity in eosinophils and airway epithelium. Photomicrographs (x400) 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 = 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 (x200).

 
To analyze epithelial CCR3 expression in vitro, we stimulated BEAS-2B (Fig. 2GoA) and PBEC (n = 2, not shown) for 24 h with TNF-{alpha} (100 ng/ml) and found induction of CCR3 mRNA by Northern blot analysis. Treatment of cells with IFN-{gamma} for 24 h did not induce CCR3 mRNA; however, IFN-{gamma} potentiated TNF-{alpha}-induced CCR3 mRNA expression, which increased under such stimulation in a concentration-dependent fashion (Fig. 2GoB). In two experiments, incubation with IL-4 (50 ng/ml) or IL-10 (10 ng/ml) for 24 h did not induce CCR3 mRNA. IL-4 partially inhibited TNF-{alpha}-induced CCR3 expression up to 39%, while IL-10 modestly up-regulated TNF-{alpha}-induced CCR3 up to 30% (p = NS, not shown). CCR3 mRNA was weakly detected in unstimulated BEAS-2B cells in one of three experiments. In contrast with mRNA expression, the levels of CCR3 protein detected by Western blot analysis of resting and TNF-{alpha} plus IFN-{gamma}-stimulated BEAS-2B cells (Fig. 2GoC) 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 = 1.55 ± 0.13, n = 11) and was slightly increased by stimulation with TNF-{alpha} plus IFN-{gamma} (MFI fold control = 1.69 ± 0.19, n = 10). Unstimulated 16-HBE cells (MFI fold control = 1.70 ± 0.13, n = 12) and PBECs (MFI fold control = 1.52 ± 0.16, n = 3) expressed similar levels of CCR3 (Fig. 2GoD). Epithelial CCR3 levels were considerably less than on eosinophils from allergic donors (MFI fold control = 11.6 ± 1.4, n = 15; not shown).



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  		FIGURE 2. Detection of CCR3 in BEAS-2B cells. A, Northern blot analysis of BEAS-2B cells treated with control medium, TNF-{alpha} (100 ng/ml) or IFN-{gamma} (100 ng/ml) alone or in combination for 24 h. I, Autoradiography of CCR3 mRNA expression (1.6 kb; representative of n = 5); II, control GAPDH mRNA expression. B, Densitometric analysis of Northern blot assay showing increasing expression of CCR3 mRNA in BEAS-2B cells upon stimulation with increasing concentrations of TNF-{alpha} plus IFN-{gamma} (n = 4; *, p < 0.05 compared with unstimulated cells). C, Western blot analysis (representative of n = 3), performed using the same Ab used for immunohistochemistry (7B11). Identical results were obtained when using the polyclonal Ab, H-52 (n = 2, data not shown). Purified (>98% purity) human eosinophils (Eos) or neutrophils (Neuts) were used as positive and negative controls, respectively. D, Surface expression of CCR3 on PBEC (top; representative of n = 3) and BEAS-2B (bottom; representative of n = 11). Histograms of flow cytometric analysis with anti-CCR3 (7B11) are represented by the dark line histograms and the IgG2a isotype control are depicted by the light line histograms.

 
In two of five experiments, a sharp increase in intracellular Ca2+ was observed in unstimulated BEAS-2B cells loaded with fura-2 and challenged with the CCR3 agonist, eotaxin (10 nM) and was similar to that seen with bradykinin, which also binds to a seven-transmembrane G protein-coupled receptor (Fig. 3GoA). Eotaxin stimulation also induced tyrosine phosphorylation of cellular proteins including members of the mitogen-activated protein kinase family, which was maximal at 0.5 min (Fig. 3GoB; n = 3 primary cells and n = 3 in 16-HBE (not shown)). These signaling events were inhibited by either pretreatment with 7B11 or pertussis toxin (n = 3 primary cells and n = 3 in 16-HBE (not shown)).



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  		FIGURE 3. CCR3 functional assays. A, A sharp intracellular Ca2+ flux was noted in BEAS-2B cells in response to eotaxin and bradykinin (inset; positive control). B, Western blot analysis demonstrating the kinetics of phosphorylation of the p42 mitogen-activated protein kinase in eotaxin-stimulated PBECs. PBECs were preincubated with media alone or pertussis toxin (1 µg/ml, 1 h) and stimulated with eotaxin (100 ng/ml) for up to 1 min. Similar results were noted when lysates were analyzed by immunoblot for eotaxin-induced tyrosine phosphorylation with and without preincubation with the CCR3-blocking mAb 7B11. (These are representative blots of n = 3 for primary epithelial cells and n = 3 for 16-HBE cells.) To ensure equal loading in each lane, we load each lane with 10 µg of total protein (measured by the bicinchoninic acid assay) and run a parallel immunoblot probed with p42/p44 anti-ERK Ab (data not shown).

 
In competition binding assays, cold eotaxin potently inhibited the binding of 125I-eotaxin binding to BEAS-2B cells (Fig. 4Go). The C-C chemokine MIP-1{alpha}, which does not bind to CCR3, and the CXC chemokine IL-8 did not displace radiolabeled eotaxin binding even at high concentrations (100 nM).



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  		FIGURE 4. Competitive inhibition of 125I-eotaxin binding to BEAS-2B cells. BEAS-2B cells (representative of n = 2) were incubated with 0.3 nM of 125I-eotaxin in the absence ({circ}) or presence of 50 pM to 1 mM of unlabeled eotaxin (•) or 100 nM IL-8 ({square}) or MIP-1{alpha} ({blacksquare}). After 90 min at 4°C, cells were washed and radioactivity counted as described in Materials and Methods.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Chemokines and their receptors mediate the selective recruitment of leukocytes into inflammatory tissues. For example, the eosinophil-rich infiltrate seen in allergic diseases is due in part to the effects of CCR3 agonists on eosinophils. In this paper, we report that 1) human airway epithelial cells express cell surface CCR3, 2) this expression can be modulated by inflammatory cytokines, and 3) epithelial CCR3 transduces intracellular signals.

We noted concordant expression in the airway epithelium of CCR3 with two of its ligands, eotaxin and RANTES. This suggests that in vivo, CCR3 is dynamically regulated and that the expression of CCR3 and its ligands share some common regulatory elements. In vitro analysis of mRNA expression confirms this hypothesis, because CCR3 mRNA expression was modulated by TNF-{alpha} and IFN-{gamma}, which also induces eotaxin and RANTES mRNA expression in epithelial cells (13). Despite the increase of CCR3 mRNA induced by these stimuli, levels of CCR3 protein on the cell surface and from total cell lysates did not appear to be significantly increased. The reason for this discrepancy is yet to be established, but may be explained by enhanced receptor turnover and degradation (24) in the presence of its endogenous ligands. Alternatively, posttranscriptional regulation or existence of receptor-activity modifying protein-like proteins necessary for expression of mature membrane protein (25) may exist. The coexpression of functional CCR3 and some of its ligands in airway epithelium suggest that epithelial CCR3 may be involved in the regulation of the mucosal chemokine network by enabling epithelial cells to respond in an autoregulatory or juxtaregulatory fashion to CCR3 ligands.

Epithelial CCR3 is a functional G protein-coupled receptor and not merely a decoy receptor. Stimulation of both primary and immortalized epithelial cells with the CCR3-specific ligand, eotaxin, induced an intracellular Ca2+ flux and tyrosine phosphorylation. The latter function was inhibited by pertussis toxin or 7B11.

Expression of a functional CCR3 on epithelial cells indicates that the biological effects of the C-C chemokines in the airways may extend beyond migration and activation of hemopoietic cells. C-C chemokines may modulate several aspects of epithelial function, including cell migration, activation, proliferation, and apoptosis (10, 11, 26). We are exploring whether CCR3 ligands mediate 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 intracytoplasmic 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 Ca2+ signaling studies.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI 01226 and AI 44242-01. Back

2 Address correspondence and reprint request to Dr. Lisa A. Beck, Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224-6801. Back

3 Abbreviations used in this paper: MCP, monocyte chemoattractant protein; ERK, extracellular signal-regulated kinase; NHBE, normal human bronchial epithelial cells; PBEC, primary bronchial epithelial cells; MIP, macrophage inflammatory protein; MFI, mean fluorescence intensity. Back

Received for publication December 13, 1999. Accepted for publication December 8, 2000.


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 Introduction
 Materials and Methods
 Results
 Discussion
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