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Published ahead of print on May 14, 2003, doi:10.1165/rcmb.2002-0272OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 627-633, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0272OC

CCL20 Is an Inducible Product of Human Airway Epithelia with Innate Immune Properties

Timothy D. Starner, Carrie K. Barker, Hong Peng Jia, Yubin Kang and Paul B. McCray, Jr.

Department of Pediatrics, The University of Iowa College of Medicine, Iowa City, Iowa

Address correspondence to: Paul B. McCray Jr., M.D., Department of Pediatrics, University of Iowa Hospitals and Clinics, 200 Hawkins Drive, Iowa City, IA 52242. E-mail: paul-mccray{at}uiowa.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chemokine ligand 20 (CCL20) and human ß-defensins (HBDs) share structural and functional properties, including antiparallel ß-pleated sheet core structures, charge distribution, and signaling to adaptive immune cells via the highly selective CCR6 receptor. Because of their similarities, we hypothesized that in addition to its known adaptive immune signaling functions, CCL20 has antimicrobial properties and participates in pulmonary innate immunity. We found that primary cultures of human airway epithelial and cultured fetal lung explants expressed CCL20 mRNA. Expression of CCL20 transcripts were significantly induced by interleukin (IL)-1ß and tumor necrosis factor-{alpha}, and inhibited by dexamethasone. Primary cultures of airway epithelia secreted CCL20 both apically and basolaterally, and CCL20 abundance was increased over 30-fold with IL-1ß stimulation, achieving an estimated concentration of 167 ng/ml in airway surface liquid. CCL20 abundance in bronchoalveolar lavage fluid from patients with cystic fibrosis was nearly 90-fold higher compared with bronchoalveolar lavage fluid from healthy volunteers. Interestingly, CCL20 exhibited salt-sensitive antimicrobial activity, mainly against Gram-negative bacteria in low µg/ml concentrations. Additionally, apical washings from IL-1ß–stimulated primary cultures of human airway epithelia had significantly more antimicrobial activity than unstimulated controls. CCL20 rapidly permeabilized bacterial membranes with a time course intermediate to HBD-2 and HBD-3. Thus, CCL20 is a bi-functional peptide with both innate and adaptive immune properties that is regulated by inflammatory mediators, expressed by airway epithelia, and increased in cystic fibrosis airway secretions.

Abbreviations: airway surface liquid, ASL • bronchoalveolar lavage fluid, BALF • chemokine ligand 20, CCL20 • chemokine receptor 6, CCR6 • cystic fibrosis, CF • enzyme-linked immunosorbent assay, ELISA • glyceraldehyde-3-phosphate dehydrogenase, GAPDH • human ß-defensin, HBD • interferon-{gamma}, IFN-{gamma} • interleukin, IL • minimal inhibitory concentration, MIC • o-nitrophenyl-ß-D-galactopyranoside, ONPG • polymerase chain reaction, PCR • reverse transcription, RT • tumor necrosis factor-{alpha}, TNF-{alpha}


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Chemokine ligand 20 (CCL20), also known as liver and activation-regulated chemokine (LARC), Exodus-1, macrophage inflammatory protein-3{alpha} (MIP-3{alpha}), and small inducible cytokine subfamily A member 20 (SCYA20), is a chemokine discovered through bioinformatic technologies (1, 2). It stimulates the migration of B-cells (3), immature dendritic cells (4), and a subset of memory T cells (5) via the chemokine receptor 6 (CCR6) receptor. CCL20 has specific high affinity for CCR6 (4) and is the only known chemokine ligand for CCR6 (6). The structural basis for the highly specific interactions between CCL20 and CCR6 is not fully elucidated, but may involve the N-terminal Asp-Cys-Cys-Leu (DCCL) motif and the narrow groove formed by the N-terminal loop and the turn between ß2 and ß3 on CCL20 (7). Interestingly, two cationic antimicrobial peptides involved in mucosal innate immunity, human ß-defensin (HBD)-1 and HBD-2, have also been shown to stimulate memory T cell and immature dendritic cell migration via the highly selective CCR6 receptor (8). Although they share little primary sequence homology, CCL20 and the ß-defensins share a high degree of structural homology. Both have a similar three ß-pleated sheet core structure stabilized by dicysteine bonds, similar distribution of positively charged amino acids, and similar N-terminal motifs (7). Structurally, CCL20 can be thought of as larger version of a ß-defensin with extended C- and N-terminal groups.

There are other similarities between CCL20 and the ß-defensins. Epithelia express both peptides (9–14). The inflammatory cytokines interleukin (IL)-1ß and tumor necrosis factor-{alpha} (TNF-{alpha}) upregulate expression of both peptides (9, 10, 13) via the nuclear factor (NF)-{kappa}B pathway (12, 15). Airway epithelia produce ß-defensins (13), and CCL20 mRNA has been detected in human lung tissue by Northern blot analysis (1, 2), but to our knowledge, the cellular origin of CCL20 is not defined, and CCL20 protein production by pulmonary cells has not been investigated in in vitro or in vivo systems. Another potential similarity is antimicrobial activity. ß-defensins are best known for their antimicrobial activity (16), and recently members of the related interferon (IFN)-inducible ELR-CXC cytokines were shown to exhibit antimicrobial activity (17).

Because of the similarities between CCL20 and the ß-defensins, we hypothesized that CCL20 is produced by airway epithelia, is regulated by inflammatory mediators, and possesses innate antimicrobial properties. We found that airway epithelia inducibly express CCL20 and that the protein was secreted in a polar manner with 8-fold higher estimated apical concentrations. CCL20 abundance was significantly higher in bronchoalveolar lavage fluid (BALF) from patients with cystic fibrosis (CF) compared with BALF from healthy volunteers. Importantly, CCL20 displayed salt-sensitive antimicrobial activity against mainly Gram-negative bacteria and permeabilized bacterial membranes in a range intermediate to HBD-2 and HBD-3.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
Recombinant CCL20, HBD-2, and HBD-3 peptides (Peprotech, Rocky Hill, NJ) were analyzed for purity and concentration using mass spectroscopy and amino acid composition. The human cathelicidin, LL37, was generously provided by Dr. Robert Lehrer (UCLA). Peptides were stored in 0.02% acetic acid at –80°C. Before use in antimicrobial assays, peptides were lyophilized and then reconstituted in 0.02% acetic acid with 0.1% human serum albumin.

Culture Models and Human Samples
Primary cultures of human airway epithelia. Primary cultures of well differentiated human airway epithelia were prepared and grown at the air–liquid interface as previously described (18). In cytokine stimulation experiments, epithelia were incubated overnight at 37°C in 5% CO2 with 100 µl of phosphate-buffered saline apically and 500 µl of media basolaterally. Stimulated apical and basolateral fluids additionally contained 100 ng/ml of recombinant human interlukin-1ß (IL-1ß) (Sigma, St. Louis, MO), 100 ng/ml IFN-{gamma} (Sigma), 100 ng/ml both IL-1ß and IFN-{gamma}, 10-7 M dexamethasone (Roxanne, Columbus, OH), or 100 ng/ml tumor necrosis factor {alpha} (TNF-{alpha}; Sigma). For assays of the antimicrobial activity of apical washings, primary cultures were incubated and repeatedly washed in antibiotic-free media for 5 d to remove any residual antibiotics. Apical washings for the antimicrobial assay were obtained after 24 h of cytokine stimulation by adding 100 µl of sterile water to the apical surface and immediately pipetting off the apical fluid.

Human fetal lung explant cultures. Mid-gestational fetal lung explants were cultured as previously described (19). Briefly, tissues were dissected free of major airways and blood vessels, cut into 1- to 2-mm pieces, and cultured at 37°C in serum and hormone free Waymouth's MB752/1 medium (Gibco, Grand Island, NY) supplemented with 1% Penicillin/Streptomycin (Gibco 15140–122) and 0.1% Amphotericin B (Cellgro 30–003-C1, Herndon, VA) in a humidified atmosphere with 5% CO2. Tissues were incubated either in control medium or in medium containing 100 ng/ml of recombinant human IL-1ß, 100 ng/ml IFN-{gamma}, 100 ng/ml of both IL-1ß and IFN-{gamma}, 10-7 M dexamethasone, 106 colony-forming units of heat killed E. coli DH5{alpha} bacteria, or 100 ng/ml of phorbol-12-myristate-13-acetate (PMA; Sigma). Explants were maintained in culture for 24 h. The tissue samples were then frozen and stored at –80°C before analysis by reverse transcription (RT)-polymerase chain reaction (PCR). For each experimental condition, replicates were performed using tissues from different donors (n = 4–5).

Human bronchoalveolar lavage samples. Bronchoalveolar lavage fluid samples were obtained from 21 CF patients and 8 healthy volunteers. Samples were stored at –80°C prior to analysis by enzyme-linked immunosorbent assay (ELISA).

All BALF specimens and culture samples used in these studies were obtained using protocols approved by the Institutional Review Board at The University of Iowa.

RNA Isolation and Semiquantitative RT-PCR
Total RNA was extracted from primary cultures of airway epithelia and fetal lung explant cultures using TriReagent (Molecular Research Center, Inc., Cincinnati, OH) per manufacturer's recommendations. The mRNA samples were treated with DNase (Promega, Madison, WI) per manufacturer's specifications. RT was performed using 1 µg of RNA with Superscript II reverse transcriptase kit (Gibco) and random sequence hexamers. After treatment with Biolase DNA Polymerase (Bioline, Canton, MA), RT reaction pruducts were subjected to PCR using an automated DNA thermal cycler for 25 cycles. PCR conditions included: denaturation 30 s at 94°C; annealing 30 s at 60°C; extension for 30 s at 72°C; and a final extension at 72°C for 5 min. The following primers were used: CCL20 forward primer 5'-GCA AGC AAC TTT GAC TGC TG-3'; CCL20 reverse primer 5'-TGG GCT ATG TCC AAT TCC AT-3'; GAPDH forward primer 5'- GTC AGT GGT GGA CCT GAC CT-3'; GAPDH reverse primer 5'-AGG GGT CTA CAT GGC AAC TG-3'. PCR products were visualized after electrophoresis in 2% agarose gels and staining with ethidium bromide. The CCL20 PCR product was sequenced to confirm proper primer amplification. For analysis of the fetal lung cultures, densitometry was performed using a ChemiImager 4,400 (AlphaInnotech, San Leandro, CA). To quantify the abundance of CCL20 expression, densitometry values were standardized to their corresponding GAPDH control.

CCL20 Protein Quantification by ELISA
CCL20 protein concentration was measured by ELISA (#DM3A00; R&D Systems, Minneapolis, MN) per manufacturer's instructions. To determine the polarity of secretion, primary cultures of human airway epithelia were grown in the presence or absence of 100 ng/ml of IL-1ß as described above. Apical washings were obtained by applying 100 µl of phosphate-buffered saline onto the apical surface; the basolateral sample consisted of a 100 µl sample of the 500 µl basolateral culture media. Samples were stored at –80°C before analysis by ELISA and were run in triplicate to ensure reproducibility.

Antimicrobial Assays
We used a modified radial diffusion assay to investigate bacterial sensitivity to CCL20 as previously described (20). Briefly, 4 x 106 bacteria in mid-log phase were suspended in an underlay gel. Three millimeter diameter wells were punched into the gel and filled with peptide at concentrations ranging from 0.25–250 µg/ml or 0.0085–8.5 µl of the 100-µl apical washings (see PRIMARY CULTURES OF HUMAN AIRWAY EPITHELIA above) that were lyophilized and then reconstituted in 0.02% acetic acid with 0.1% human serum albumin. The plates were then incubated for 3 h at 37°C. Nutrient-rich gels were then overlaid and the plates were incubated overnight at 37°C. Zones of clearance were manually measured and plotted on a semi-log graph where the x-intercept represents the minimal inhibitory concentration (MIC). Low salt agarose underlay gels contained 10 mM sodium phosphate (pH 7.4), whereas high salt underlay gels contained 150 mM sodium chloride (pH 7.4). Test organisms included Candida albicans ATCC 1023, Escherichia coli DH5{alpha}, E. coli ML35p, Klebsiella pneumoniae ATCC 13883, Moraxella catarrhalis ATCC 25238, Pseudomonas aeruginosa PA01, Staphylococcus aureus ATCC 29213, Staphylococcus epidermidis ATCC 12228, and clinical strains of Enterococcus species, Group B Streptococcus, Listeria monocytogenes, methicillin-sensitive Staphylococcus aureus, and mucoid Pseudomonas aeruginosa.

Permeability of Bacterial Membranes
Permeabilization of bacterial membranes was measured using E. coli ML35p expressing an intracellular ß-galactosidase reporter as previously described (21). Briefly, 106 colony-forming units of E. coli ML35p were incubated with 2-fold dilutions of peptide ranging from 8–0.13 µg/ml diluted in 10 mM sodium phosphate with 3 mM o-nitrophenyl-ß-D-galactopyranoside (ONPG; Sigma) at 37°C in a 96-well plate. Samples were immediately analyzed at 405 nm every minute for 1 h using a VERSAmax microplate reader (Molecular Devices, Sunnyvale, CA). Peptides tested included HBD-2, HBD-3, CCL20, and the human cathelicidin LL37. Negative controls consisted of the 10 mM sodium phosphate, 3 mM ONPG diluent without peptide. These studies were performed in triplicate.

Statistical Analysis
All analyses of statistical significance were performed with Student's t test using Microsoft Excel. P values < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulated Expression of CCL20 mRNA in Human Airway Epithelia and Fetal Lung Explants
We investigated the expression and regulation of CCL20 transcripts in primary cultures of well-differentiated human airway epithelia and fetal lung explants using semiquantitative RT-PCR. As shown in Figure 1, airway epithelia predominantly express CCL20 in the IL-1ß– and TNF-{alpha}–stimulated conditions.



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  		Figure 1. Airway epithelia inducibly express CCL20. RNA was extracted from primary cultures of airway epithelia with and without stimulation as indicated. (A) Average densitometry measurements for CCL20 mRNA standardized to GAPDH were compared with the media control condition. (B) Representative RT-PCR results for the corresponding GAPDH and CCL20. (n = 3). *P < 0.05.

 
To further address the regulation of CCL20 mRNA expression, we used mid-gestational fetal lung explant cultures. As shown in Figure 2, we detected no CCL20 message in the start tissue before culturing. After 1 d of growth in serum- and hormone-free media, the CCL20 message significantly increased (P = 0.0001). Tissues stimulated with 100 ng/ml of IL-1ß, or a combination of IL-1ß and IFN-{gamma}, demonstrated over 2-fold greater signal than media alone (P = 0.03 and P = 0.002, respectively). However, as in the human airway epithelial cultures, there was no additive increase in the combined IL-1ß and IFN-{gamma} signal over that of IL-1ß alone. Similarly, compared with media controls, there was no significant difference in response to IFN-{gamma} stimulation alone. These results suggest that IFN-{gamma} does not upregulate CCL20 expression. Treatment with the anti-inflammatory corticosteroid dexamethasone significantly decreased CCL20 message (P = 0.002). Treatment with heat-killed E. coli or PMA caused no significant change in CCL20 mRNA.



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  		Figure 2. Inflammatory mediators regulate CCL20 expression. RNA was extracted from stimulated fetal lung explant samples. Tissues were cultured overnight with and without stimulation as indicated. (A) Average densitometry measurements for CCL20 mRNA standardized to GAPDH were compared with the media control condition (n = 4–5). (B) Representative RT-PCR results for the corresponding GAPDH and CCL20. Start, tissue before culturing; CTRL, media without cytokines; IL/IFN, IL-1ß and IFN-{gamma}; Dex, dexamethasone; E. coli, heat-killed E. coli.

 
CCL20 Is Secreted by Airway Epithelial Cells and Is Present in BALF
To correlate CCL20 mRNA expression with protein abundance, we performed ELISA for CCL20 on samples from airway epithelial cultures and BALF. As shown in Figure 3, there were low levels of CCL20 in the unstimulated apical samples from airway epithelia, and CCL20 abundance increased 40-fold following IL-1ß stimulation for 24 h (P < 0.05). The mean total CCL20 secreted apically and basolaterally were 0.2 ng and 10.6 ng, respectively. Because the depth of the airway surface liquid (ASL) in cultures grown at the air–liquid interface is ~ 20 µm (22) and the surface area of the epithelial sheet is 0.6 cm2, we estimate an average apical CCL20 concentration of 167 ng/ml, with a range from 27–513 ng/ml.



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  		Figure 3. Polar secretion of CCL20. Estimated CCL20 concentrations in apical and basolateral samples from primary cultures of airway epithelia grown at the air-fluid interface, with or without 100 ng/ml IL-1ß stimulation. CCL20 peptide abundance was measured by ELISA.

 
The mean 10.6 ng of CCL20 secreted basolaterally is distributed in the 500 µl of basolateral media, resulting in an average concentration of 21.2 ng/ml with a range of 13.6–28.0 ng/ml. Similar to the apical condition, there was a 30-fold increase in basolateral CCL20 abundance following IL-1ß stimulation (P = 0.0001). Thus, the estimated apical concentrations of CCL20 are 8-fold higher than the basolateral concentrations. These results are consistent with the RT-PCR findings in Figure 1, confirming that airway epithelia secrete CCL20 and protein abundance increases following IL-1ß stimulation.

To correlate in vitro protein measurements of CCL20 with in vivo physiology, we determined CCL20 protein abundance in BALF samples from healthy adults and patients with CF. A hallmark of CF lung disease is the presence of chronic inflammation and inflammatory mediators, such as IL-1ß, IL-8, and TNF-{alpha} (for review see Ref. 23). We hypothesized that CCL20 expression may be increased in CF. As shown in Figure 4, the normal BALF samples contained low levels of CCL20, with a mean of 10.4 pg/ml. In contrast, CF samples averaged nearly 90-fold higher with a mean of 907 pg/ml (P < 0.001). The CCL20 levels in the CF BALF varied considerably among different patients, ranging from levels of the normal controls to nearly 500 times higher. Determination of the correct dilution factor for the saline infused to obtain ASL in BALF samples is controversial, but is estimated to be ~ 1 ml ASL per 100 ml BALF based on urea concentration measurements (24). Using this 100-fold dilution estimate, the CCL20 protein levels in the ASL of CF samples would average 90 ng/ml and range to a high of 463 ng/ml. These results are consistent with the estimated concentration of CCL20 in the apical ASL from primary cultures of airway epithelia previously mentioned.



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  		Figure 4. CCL20 is elevated in CF BALF samples. CCL20 abundance in BALF from normal subjects and from patients with CF was measured by ELISA. Normal BALF had very low levels of CCL20, mean 10.4 pg/ml. CF BALF exhibited nearly a 90-fold increase in CCL20, mean 907 pg/ml (P < 0.001).

 
We also investigated whether human airway epithelia express the CCR6 receptor. We were unable to detect CCR6 mRNA in control or IL-1ß–stimulated primary cultures of airway epithelia or fetal lung explants after 35 cycles of RT-PCR (data not shown). Similarly, we detected no CCR6 protein by FACS analysis or immunohistochemistry (data not shown). These results indicate that although CCL20 is secreted by airway epithelia, it acts on targets other than the epithelial cell.

CCL20 Exhibits Salt-Sensitive Antimicrobial Activity
We tested the antimicrobial activity of CCL20 against fourteen organisms, including Gram-negative and Gram-positive bacteria, and a fungus. We also determined whether the antimicrobial activity of CCL20 was salt-sensitive, as has been shown with the ß-defensins (13). As shown in Table 1, CCL20 killed six bacteria in low µg/ml concentrations, including E. coli DH5{alpha}, E. coli ML35p, K. pneumoniae, L. monocytogenes, M. catarrhalis, and P. aeruginosa PA01. The MIC of the clinical mucoid strain of P. aeruginosa was relatively low, but was four times higher than the nonmucoid P. aeruginosa PA01. CCL20 exhibited poor antimicrobial activity against C. albicans, Group B Streptococcus, S. aureus, and S. epidermidis, and no activity against a clinical strain of methicillin-sensitive S. aureus and two clinical strains of Enterococcus species. Thus, CCL20 killed all of the Gram-negative bacteria tested, and showed poor activity against all of the Gram-positive bacterium, with the exception of L. monocytogenes. As with the ß-defensins, the antimicrobial activity of CCL20 was markedly inhibited by isotonic salt concentrations in all three strains of bacteria tested.


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  		TABLE 1 CCL20 exhibits salt-sensitive antimicrobial activity

 
IL-1ß Stimulated ASL has Increased Antimicrobial Activity
We tested the antimicrobial activity of apical washings from control and IL-1ß–stimulated primary cultures of airway epithelia grown in antibiotic-free media (Figure 5). Both stimulated and unstimulated washings exhibited potent antimicrobial activity, but the washings from IL-1ß–stimulated cells displayed a 2.6-fold increase in antimicrobial activity (P < 0.05) against E. coli DH5{alpha} as measured by radial diffusion assay. As an additional control, 100 ng/ml of IL-1ß was tested by radial diffusion assay and had no antimicrobial activity.



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  		Figure 5. IL-1ß–stimulated ASL has increased antimicrobial activity. Antimicrobial activity of washings from primary cultures of airway epithelia either with or without IL-1ß stimulation. Data shown as fold increase from media control (n = 3). *P < 0.05.

 
CCL20 Permeabilizes Bacterial Membranes
To determine the effects of CCL20 on bacterial membrane permeability, we used an E. coli ML35p ß-galactosidase assay (21). An increase in absorbance at 405 nm in this assay indicates permeability of both inner and outer membranes, allowing extracellular ONGP to enter the bacteria and become a substrate for the intracellular ß-galactosidase. Figure 6 shows a representative experiment using an 8 µg/ml peptide concentration. All peptides displayed a dose–response on bacterial membrane permeability, with decreasing permeability seen in the serial dilutions from the 8 µg/ml concentration downward (data not shown). CCL20 rapidly permeabilized bacterial membranes with a time course intermediate to HBD-2 and HBD-3. It is notable that the pattern and time course of permeability for CCL20 are closer to those seen in HBD-2. For CCL20 and HBD-2, the permeability signal began to rise at ~ 10 min and gradually increased over time. In contrast, HBD-3 and LL37 began to permeabilize bacterial membranes within 1–3 min, and permeability increased more rapidly. Thus, these data show that CCL20 efficiently permeabilizes E. coli ML35p bacterial membranes.



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  		Figure 6. CCL20 permeabilizes bacterial membranes. Permeability of E. coli ML35p membranes as measured by absorbance at 405 nm. A representative graph of the 8 µg/ml peptide concentrations is shown (n = 3).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These are the first studies to demonstrate that CCL20 is an inducible product of human airway epithelia. Furthermore, CCL20 is secreted in a polar manner and its expression is increased in CF airway secretions. In addition to its well-documented chemotaxis functions, we show that CCL20 exhibits a specific spectrum of salt-sensitive antimicrobial activity, especially against Gram-negative organisms. It also permeabilized bacterial membranes, suggesting one possible mechanism for its antimicrobial properties.

We demonstrated that lung tissues express CCL20 mRNA. This is in agreement with previous studies (1, 2). Importantly, our studies in primary cultures show that airway epithelial cells abundantly express CCL20. We showed that lung epithelia inducibly express CCL20 mRNA with IL-1ß and TNF-{alpha} stimulation, and additionally that there was a corresponding increase in CCL20 protein abundance with IL-1ß stimulation. CCL20 expression has also been shown to be increased by IL-1ß stimulation in intestinal epithelia (25) and skin keratinocytes (9). We also found that corticosteroid treatment inhibited CCL20 mRNA in the fetal lung explant model. The anti-inflammatory cytokine IL-10 has also been shown to downregulate CCL20 message (1). We did not see significant inhibition from dexamethasone in the primary cultures of human airway epithelia; however, the low basal expression of CCL20 mRNA in the control would make it difficult to show inhibition with this model. The significant increase in CCL20 message after the start tissue has been cultured for 24 h in the fetal lung explant model (Figure 2) supports the notion that CCL20 expression is developmentally regulated. However, its induction could also be an in vitro cell culture phenomenon or response to injury from tissue manipulation. The fetal lung explants may not be the best model to investigate induction of CCL20 by heat-killed bacteria because the bacterial products may not be able to adequately penetrate into the cultured tissue. Furthermore, CCL20 induction may use pathways that do not involve heat-killed bacterial products or PMA.

Airway epithelia secrete CCL20 in a polar fashion. The estimated concentration in the IL-1ß–stimulated apical fluid is 8-fold higher than in the basolateral fluid in this culture model. Although there is a greater total amount of CCL20 secreted basolaterally, the apically secreted protein is concentrated in the small volume of ASL. The apically secreted 0.2 ng is diluted in 1.2 µl of ASL, resulting in an estimated apical concentration of 167 ng/ml. In contrast, the basolateral 10.6 ng is distributed in 500 µl of media, resulting in an overall concentration of 21.2 ng/ml. Because it is difficult to estimate the basolateral volume of distribution, the in vivo concentration of CCL20 in the subepithelial space cannot be precisely determined. However, we speculate that secretion of CCL20 in the airway would create a gradient with the highest concentrations in the ASL or immediate basolateral area. This would effectively create a CCL20 concentration gradient that could attract cells of the adaptive immune system to migrate toward the lumen of the airway. Importantly, the average apical concentration of CCL20 in this model is within the ~ 100 ng/ml level reported to induce maximal lymphocyte cell migration (8).

On average, CF BALF contained nearly 90-fold higher concentrations of CCL20 than the BALF of healthy volunteers, which were at the lower limit of detection. The increased abundance of cytokines such as IL-1ß, TNF{alpha}, and IL-8 in CF ASL (26, 27) may stimulate the production of CCL20 by airway epithelia. We also measured CCL20 abundance in one BALF from a patient with chronic granulomatous disease and CCL20 was similarly elevated (109 ng/ml). However, caution should be taken in interpreting the source of CCL20 detected in the BALF. Although CCL20 may be apically secreted by airway epithelial cells, it is also produced by neutrophils (28). There may also be contributions from other cells in the airway that have not yet been identified. However, given the inducible expression of CCL20 message and protein, the airway epithelia likely represent a significant source of the CCL20 detected in the CF BALF. As with the results from IL-1ß–stimulated airway epithelial cultures, the concentrations of CCL20 measured in the CF BALF resulted in an average estimated CCL20 concentration in the ASL that was in the maximal range for stimulating lymphocytic cell migration (8). Because the CF airway submucosa has significantly increased numbers of T cells and B cells (29), CCL20 may be one factor inducing lymphocyte recruitment. Increased numbers of lymphocytes may be participating in the inflammatory responses in CF that lead to destruction of airway tissue and the progression of lung disease.

The finding that CCL20 kills mainly Gram-negative bacteria in a salt-sensitive manner may have clinical implications, because CCL20 exhibits antimicrobial activity against many lung pathogens. The antimicrobial activities shown for CCL20 (Table 1) are generally in agreement with those of Hoover and coworkers, who recently reported that CCL20 has antimicrobial activity in a low µg/ml range against E. coli but not S. aureus (30). Although the levels of CCL20 detected in both the IL-1ß–stimulated airway epithelial cultures and CF BALF are below the MICs for the bactericidal activity, CCL20 could act in concert with numerous other antimicrobial factors in ASL, such as lactoferrin, lysozyme, secretory leukocyte protease inhibitor, and the ß-defensins (31). CCL20 might exhibit synergistic antimicrobial activity in a manner similar to that seen between lysozyme, lactoferrin, and secretory leukocyte protease inhibitor (32). Also, there may be microenvironments that have much higher concentrations of CCL20, such as ASL, the paracellular space, or directly under the basolateral membrane. CCL20 may be in high enough concentrations in these microenvironments to exert direct antimicrobial activity. Furthermore, the IL-1ß–stimulated airway washings exhibited significantly more antimicrobial activity than unstimulated washings (Figure 5). Although there are many antimicrobial factors in addition to CCL20 that were likely induced by IL-1ß, CCL20 likely contributed to this increase in ASL microbicidal activity. Additionally, the MICs we measured for CCL20 are generally analogous to those reported for the ß-defensins and other antimicrobial peptides of the innate immune system (10, 11, 17, 20).

CCL20 permeabilized bacterial membranes at the same concentration and with a similar time course as the ß-defensins. Because defensins are thought to kill bacteria by disrupting bacterial membranes (21, 33), the antimicrobial activity of CCL20 may be similarly mediated by cell membrane permeabilization. HBD-3 and LL37 caused a greater and more rapid increase in permeability than did HBD-2 or CCL20. These differences in permeability may reflect differences in binding sites or even mechanisms of action.

In conclusion, CCL20 is a bi-functional peptide in airway host defense, participating in both the innate and adaptive immune systems. It is secreted both apically and basolaterally by airway epithelia in response to inflammatory stimuli, and exerts antimicrobial activity against a wide spectrum of mainly Gram-negative bacteria. If invading organisms are not quickly eliminated by CCL20 and other antimicrobial factors of the innate immune system, the CCL20 chemokine gradient may elicit the migration of CCR6+ B cells, T cells, and immature dendritic cells to the site of invasion and thereby initiate an adaptive immune response.


   Acknowledgments
 
These studies were supported in part by grants from the National Institutes of Health (HL61234, P.B.M.; HL07638 and HL67992-01, T.D.S). The authors acknowledge the support of the Cell Culture Core, partially supported bye the Center for Gene Therapy for Cystic Fibrosis (NIH P30 DK-54759) and the Cystic Fibrosis Foundation, for preparing airway epithelial cultures. The authors thank Dr. Michael Selsted for providing E. coli ML35p and technical assistance with the permeability assay, and Dr. Michael Acarregui for assistance with the fetal lung explant cultures. They thank Jian Q. Shao for his technical assistance. They also thank Joe Zabner and Jerrold Weiss for providing critical commentary on the manuscript.

Received in original form November 25, 2002

Received in final form May 2, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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