CCL20, like human β-defensin (hBD)-2, is a potent chemoattractant for CCR6-positive immature dendritic cells and T cells in addition to recently found antimicrobial activities. We previously demonstrated that IL-17 is the most potent cytokine to induce an apical secretion and expression of hBD-2 by human airway epithelial cells, and the induction is JAK/NF-κB-dependent. Similar to hBD-2, IL-17 also induced CCL20 expression, but the nature of the induction has not been elucidated. Compared with a panel of cytokines (IL-1α, 1β, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 18, IFN-γ, GM-CSF, and TNF-α), IL-17 was as potent as IL-1α, 1β, and TNF-α, with a time- and dose-dependent phenomenon in stimulating CCL20 expression in both well-differentiated primary human and mouse airway epithelial cell culture systems. The stimulation was largely dependent on the treatment of polarized epithelial cultures from the basolateral side with IL-17, achieving an estimated 4- to 10-fold stimulation at both message and protein levels. More than 90% of induced CCL20 secretion was toward the basolateral compartment (23.02 ± 1.11 ng/chamber/day/basolateral vs 1.82 ± 0.82 ng/chamber/day/apical). Actinomycin D experiments revealed that enhanced expression did not occur at mRNA stability. Inhibitor studies showed that enhanced expression was insensitive to inhibitors of JAK/STAT, p38, JNK, and PI3K signaling pathways, but sensitive to inhibitors of MEK1/2 and NF-κB activation, suggesting a MEK/NF-κB-based mechanism. These results suggest that IL-17 can coordinately up-regulate both hBD-2 and CCL20 expressions in airways through differentially JAK-dependent and -independent activations of NF-κB-based transcriptional mechanisms, respectively.
Innate immunity in respiratory tract epithelia is critical to eliminate pathogens efficiently and control infection in the lung. Respiratory tract epithelia, in addition to providing an anatomic barrier, secrete a variety of peptides, such as β-defensins and chemokines, which act either directly or indirectly against invading pathogens (1). The chemokines are a group of small (<15 kDa) inducible, proinflammatory chemoattractant cytokines that interact with leukocytes via specific receptors to elicit the departure of leukocytes from the circulation and the infiltration to tissues at sites of immune challenge (2). Thus far, over 40 chemokines have been identified, and can be classified into four subfamilies based on the arrangement of cysteine residues (C, CC, CXC, or CX3C) (3).
CCL20 (4) or MIP-3α, also known as liver and activation-regulated chemokine and Exodus-1, is a CC chemokine that has antimicrobial activity comparable to β-defensins (5). It has been shown that CCL20 and human β-defensin-2 (hBD-2)3 share structural, functional, and regulatory properties (6). In a structural sense, they both include antiparallel β-sheet core structures and the charge distribution is similar. CCL20 has been demonstrated in tracheobronchial airways (4). CCL20 is up-regulated by proinflammatory stimuli, such as IL-1β, TNF-α, and LPS (7, 8, 9, 10, 11, 12). CCL20 is the only chemokine known to interact with CCR6, a property also shared with the antimicrobial β-defensins (13). CCR6 has an important role in mediating dendritic cell localization and lymphocyte homeostasis in mucosal tissues (14). It has been suggested that through the local production of CCL20, immature dendritic cells, effector or memory CD4+ T lymphocytes, and B lymphocytes migrate to the site of inflammation to encounter the invading pathogens (15, 16). In airway epithelium, CCL20 expression by airway epithelium is inducible by TNF-α, IL-1β, and Th2 cytokines (6, 17), and the level of CCL20 is significantly higher in bronchoalveolar lavage fluid (BALF) from patients with cystic fibrosis compared with BALF from healthy volunteers (6).
Our recent work established that IL-17-stimulated mucin genes MUC5AC and MUC5B (18) and hBD-2 (19) expressions in well-differentiated primary human tracheobronchial epithelial (TBE) cells. Through a gene expression profile analysis, we have also observed a similar stimulation of CCL20 transcript by IL-17 (19). Human IL-17 (or IL-17A) is exclusively expressed by activated CD4+ and CD8+ T memory cells, primarily of the prototypic CD45+RO+ subtype. In mouse lung, IL-17 is produced by CD4+ and CD8+ cells through IL-23-mediated and TLR4-dependent pathways (20). IL-17 stimulates the expression of proinflammatory and neutrophil-mobilizing cytokines, such as IL-1β, IL-6, TNF-α, IL-8, MIP-2, growth-related oncogene, granulocyte chemotactic protein 2, and GM-CSF in rodent and human cells (21, 22). IL-17 has recently been shown as an important cytokine against the microbial infection in airways, and the levels of IL-17 were also significantly elevated in BALF of infected lung diseases and in allergic asthmatic airways (23).
In this communication, we set out to examine the effects of IL-17 and its signaling pathways that are potentially involved in the stimulation of CCL20 expression by well-differentiated primary human TBE cells. Similar to hBD-2 stimulation, we have found that IL-17 is as potent as TNF-α and IL-1β in the stimulation of CCL20 expression, as compared with a panel of cytokines (IL-1α, 1β, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 18, TNF-α, IFN-γ, and GM-CSF). Moreover, we found that IL-17 may stimulate CCL20 expression via a JAK-independent but MEK/NF-κB activation-based transcriptional mechanism, when applied basally to the TBE cells. In contrast to an apical secretion of hBD-2 (19), >90% of stimulated CCL20 was basolaterally secreted by IL-17-treated TBE cells.
Materials and Methods
Recombinant human IL-1α, 1β, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 18, TNF-α, IFN-γ, GM-CSF, and goat anti-IL-17RA Ab were purchased from R&D Systems. Mouse anti-p65 mAb was purchased from Santa Cruz Biotechnology. Helenalin, LY29402, U0126, SB203580, JNK inhibitor II, actinomycin D, and JAK inhibitor I were purchased from Calbiochem-Novabiochem.
Cell culture from human and mouse airway tissues
Human tracheobronchial tissues were obtained from the University of California at Davis, School of Medicine (Sacramento, CA) with patient consent and also from the National Disease Research Interchange (Philadelphia, PA). The University Human Subjects Review Committee approved all procedures involved in tissue procurement. In this study, tissues were collected only from patients without diagnosed lung-related disease or known history of airway disease. Primary cultures derived from these airway tissues have been previously established (24, 25). Briefly, protease-dissociated TBE cells were plated on Transwell chambers (25 mm, Corning; Costar) at 1–2 × 104 cells/cm2 in Ham’s F12/DMEM (1:1) supplemented with insulin (5 μg/ml), transferrin (5 μg/ml), epidermal growth factor (10 ng/ml), dexamethasone (0.1 μM), cholera toxin (10 ng/ml), bovine hypothalamus extract (15 μg/ml), BSA (0.5 mg/ml), and all-trans-retinoic acid (30 nM). After 1 wk in an immersed culture condition, the primary TBE cultures were transferred to an air-liquid interface (ALI) culture condition. At full confluence, the culture in the chamber achieved an elevated transepithelial resistance as measured using a “chopstick” voltmeter (Millicell-ERS; Millipore). Briefly, one of the chopstick electrodes was immersed to the basal side of the culture, the other electrode was immersed into the apical side of the Transwell chamber and then the transepithelial resistance was measured. By 2 wk a well-established mucociliary cell layer was developed under the ALI culture condition. At this time point, cultures were treated with IL-17 and various cytokines and inhibitors as described below. Human bronchial epithelial HBE1 cell line is an immortalized line of normal human airway epithelial cells (26). These cells were maintained in serum-free Ham’s F12 medium supplemented with six hormonal supplements, as described earlier for primary TBE cultures, but without all-trans-retinoic acid. C57BL/6 mouse tracheal epithelial cells were isolated and cultured under an ALI condition, similar to that of human primary TBE cells.
Treatments of cytokines and inhibitors of signaling pathways
Recombinant human cytokines were dissolved in PBS with 1% BSA, as suggested by the manufacturer’s protocol (R&D System). Inhibitors were dissolved in the appropriate solvent as suggested by the manufacturer’s protocol (Sigma-Aldrich). Before the treatment or media change, cytokines (0–100 ng/ml) and inhibitors were diluted to the desired levels in fresh culture media. Control culture media contained similar levels of PBS/1% BSA and/or solvent (<0.1%) as treatment media. The apical side of the Transwell chamber received 0.5 ml of the freshly made culture media containing the cytokines or inhibitors. The basal side of the chamber received 2 ml of the same. Within 5 min of the addition, the Transwell chambers were returned to ALI culture conditions in a well-humidified CO2 incubator until the next media change. Media changes were done once every other day until harvest. For the mRNA stability study, cells were pretreated with or without 20 ng/ml IL-17 for 24 h. Then, 5 μg/ml actinomycin D was added to these cultures, which were harvested for RNA isolation at various hours as indicated. JAK inhibitor I, LY29402, helenalin, U0126, SB203580, and JNK inhibitor II were dissolved in DMSO and added to both sides of the culture media 30 min before IL-17 treatment. The optimal doses for each of the selected inhibitors were determined based on the current literature and the manufacturer’s recommendations. No obvious cytotoxicity (based on trypan blue dye exclusion) was observed in these cultures after these inhibitor treatments.
Measurement of CCL20 secretion by ELISA
To determine the polarity of CCL20 secretion in culture, both sides of the Transwell chambers containing ALI cultures of primary human TBE cells were rinsed three times with fresh culture media and then treated with various doses of IL-17 as described in the study. Apical secretions were recovered by washing the apical side of the culture with 300 μl of culture media. Basolateral secretions were recovered by directly collecting the basal culture media (2 ml). After a brief centrifugation to remove cell debris and particulates, these collected washes and media were stored at −20°C before ELISA analysis. A CCL20 ELISA kit (R&D Systems) was purchased and used per manufacturer’s instructions. Samples were run in triplicate to ensure reproducibility. Three separate primary TBE cultures derived from different donors were assayed and only one set of representative results is shown.
Real-time RT-PCR expression analysis
Five μg of total RNA was reverse transcribed with Moloney murine leukemia virus-reverse transcriptase (Promega) by oligo(dT) primers for 90 min at 42°C in 20 μl of reaction mixtures and then further diluted to 100 μl with water for the subsequent procedures. Diluted cDNA (2 μl) was analyzed using 2× SYBR Green PCR Master Mix (Applied Biosystems) by an ABI 5700 or ABI PRISM 7900HT Sequence Detection System (Applied Biosystems) following the manufacturer’s protocol. Gene-specific primers were designed according to the sequences to cover the conserved peptide sequence regions. PCR primers used were: human CCL20 forward CTGGCTGCTTTGATGTCAGT, reverse CGTGTGAAGCCCACAATAAA; mouse CCL20 forward GTGGGTTTCACAAGACAGATG, reverse TTTTCACCCAGTTCTGCTTTG; human IκB-α forward CTTCAGATGCTGCCAGAGAGT, reverse GCCTCCAAACACACAGTCATC; human β-actin forward AGAAAATCTGGCACCACACC, reverse GGGGTGTTGAAGGTCTCAAA; human GAPDH forward CAATGACCCCTTCATTGACC, reverse GACAAGCTTCCCGTTCTCAG; and mouse GAPDH forward TGTGTCCGTCGTGGATCTGA, reverse CCTGCTTCACCACCTTCTTGAT. The PCR was conducted in 96-well optical reaction plates as described (19). Briefly, each well contained a 50-μl reaction mixture that contained 25 μl of the SYBR Green PCR Master mix, 1 μl of each forward and reverse primer, 21 μl of water, and 2 μl of cDNA samples. The SYBR green dye was measured at 530 nm during the extension phase. The threshold cycle (Ct) value reflects the cycle number at which the fluorescence generated within a reaction crosses a given threshold. The threshold cycle value assigned to each well thus reflects the point during the reaction at which a sufficient number of amplicons have been accumulated. The relative mRNA amount in each sample was calculated based on its threshold cycle in comparison to the threshold cycle of housekeeping genes, such as β-actin and GAPDH. The results were presented as 2(Ct of CCL20 − Ct of housekeeping gene) in arbitrary units. The purity of amplified product was determined as a single peak of dissociation curve. Real-time PCR was conducted in duplicate for each sample, and the mean value was calculated. This procedure was performed in at least two or three independent experiments.
Cloning of the CCL20 promoter-luciferase reporter plasmid
A DNA fragment containing the proximal 687 bp of the CCL20 promoter region (−626 to +61) was amplified from PstI-digested genomic DNA by PCR with the following primers: GTTGCTAGCAAATCAAGGTGAAGCTGAGGTTT (forward primer with NheI tail) and CCATAAGCTTCATGGTTTTTAGCTCAAAGAACAG (reverse primer with HindIII tail). The amplified product was subcloned into a pGL-3-basic vector (Promega) with firefly luciferase reporter gene to generate CCL20–687/Luc plasmid. The authenticity of the clone was confirmed by DNA sequencing.
Transient transfection and luciferase assay
HBE1 cells were seeded into 12-well plates at a density of 1 × 105 cells/well. One day after plating, cells were transfected with 0.5 μg of CCL20–687/Luc plasmid DNA and 50 ng of Renilla luciferase expression vector pRL-TK (Promega) using FuGENE 6-based gene transfer protocol (Roche Diagnostics) according to the manufacturer’s instruction. Eighteen hours after the transfection, cells were treated with various concentrations of IL-17, and cell extracts were prepared for reporter gene assays 24 h after the IL-17 treatment. The reporter gene assays were conducted with the Dual-Glo Luciferase Assay System (Promega) according to the manufacturer’s protocol. The relative CCL20 promoter activities were expressed as relative luciferase units after normalization to the internal control, Renilla luciferase activity. The results were averaged from triplicate wells of three separate experiments.
For IL-17RA staining, primary cells were cultured on Transwell chambers. Membranes were then mounted to a slide, washed three times with PBS for 5 min each, and then incubated with goat anti-IL-17RA polyclonal primary Abs at 1/200 dilution in blocking buffer for overnight at 4°C. Blocking buffer is made of 2% rabbit serum and 2% BSA in PBS with Tween 20 (PBST). Then samples were washed three times with PBST for 5 min each, and incubated with fluorescent-labeled Alexa Fluor 488 rabbit anti-goat IgG secondary Abs (1/500 dilution in blocking buffer) (Molecular Probes) for 1 h at room temperature. Slides were then fixed at room temperature for 30 min in PBS supplemented with 4% paraformaldehyde solution. Nuclei were counterstained with propidium iodide using VECTASHIELD mounting medium (Vector Laboratories). The staining was visualized using a Zeiss LSM 510 confocal fluorescence microscope (×60 objective).
For p65 staining, primary cells were allowed to adhere to sterile Lab-Tek II chamber slide systems (Nalge Nunc International) incubated in previously outlined experimental conditions. Cells were fixed at 4°C for overnight in PBS supplemented with 4% paraformaldehyde solution. The slide chambers were washed three times with PBS for 5 min each, permeabilized with 0.1% Triton X-100 in PBS for 30 min at 37°C, and then blocked for 30 min at 37°C. Blocking buffer is 2% rabbit serum in PBST. The samples were then stained with mouse anti-p65 monoclonal primary Abs (1/200 dilution in blocking buffer) for 1 h at 37°C, washed three times with PBST for 5 min each, and incubated with fluorescent-labeled Alexa Fluor 488 rabbit anti-mouse IgG secondary Abs (1/500 dilution in blocking buffer) (Molecular Probes) for 1 h at 37°C. Nuclei were counterstained with VECTASHIELD mounting medium with DAPI (4′,6′-diamidino-2-phenylindole; Vector Laboratories). The staining was visualized using a Zeiss AxioSkop fluorescence microscope (×40 objective).
Data are expressed as mean ± SE. The number of repetitions for each experiment is described in the results. Group differences were calculated by t test or one-way ANOVA with Bonferroni post hoc test as described in the figures. Values of p < 0.05 were considered significant.
Effect of IL-17 on the stimulation of CCL20 expression in human primary TBE cells
Real-time RT-PCR analysis was conducted for CCL20 expression in these cytokine-treated samples. As shown in Fig. 1, CCL20 message was elevated 4- to 10-fold in TBE cells by IL-1α, IL-1β, TNF-α, and IL-17. In contrast, other cytokines, especially the Th2 cytokines from 10 to 100 ng/ml, had very little influence on inducing CCL20 messages. Overnight (16 h) treatment of airway epithelial cells with IL-17 consistently resulted in a 10-fold induction of CCL20 message. This level of stimulation is the highest among the cytokine panel of the study.
Kinetics studies of IL-17 effects on CCL20 expression
The effect of IL-17 on CCL20 gene expression was dose- and time-dependent (Fig. 2). As low as 1 ng/ml IL-17 could elicit a significant stimulation of CCL20 gene expression in primary TBE cells after 48 h of treatment. Peak stimulation occurred at 20 ng/ml level, with no further increase at 100 ng/ml but mean went up compared with 20 ng/ml IL-17. Throughout this dose study, we did not observe any cytokine-induced cytotoxicity. Cell viability, as assessed by the trypan blue exclusion method, was routinely higher than 95%.
A significant level of stimulation of CCL20 message by IL-17 was seen within 3 h and an accumulating stimulation was seen at 48 h. At this time point, there was an 80-fold abundance of CCL20 messages compared with the control. To further determine whether the observed effect of IL-17 on CCL20 expression was due to a transcriptional activation, study of the effect of IL-17 on CCL20 promoter activity was conducted in HBE1 cells using a transient transfection approach with CCL20–687/Luc chimeric construct DNA. The HBE1 cell line was used because the cell line has a similar response to IL-17 for CCL20 expression (data not shown) and it is difficult to carry out gene transfection study on primary TBE cells. As shown in Fig. 2 C, there was a dose-dependent increase in the relative CCL20 promoter based luciferase activity by IL-17. At 20 and 100 ng/ml levels, IL-17 had a significant stimulation on the hBD-2 promoter activity based on ANOVA. These results further support a transcriptional mechanism of IL-17 in the regulation of CCL20 expression.
IL-17 has been shown to stabilize the mRNA of a short-lived NF-κB regulator, IκB-ζ, in NIH 3T3 cells (27). To elucidate whether IL-17 also stabilizes CCL20 mRNA, cultures treated with or without IL-17 for 24 h were exposed to the RNA synthesis inhibitor, actinomycin D. RNA were isolated at 0.5, 1, 2, 4, and 6 h after the inhibitor treatment. The stable housekeeping gene, β-actin, was chosen as the control. The relative mRNA stability of CCL20 transcript, as compared with that of β-actin, was plotted against the time of actinomycin D treatment (Fig. 3). CCL20 transcripts were as stable as β-actin mRNA in these studies, and IL-17 obviously had no influence on the half-life of the message. In contrast, the IκB-α messages, well-known short-lived mRNAs, were much less stable than β-actin in both IL-17-treated and untreated cultures after actinomycin D treatment. These results suggested that CCL20 message is as stable as β-actin in airway epithelial cultures, and the stability of CCL20 transcript is not affected by IL-17 treatment.
Stimulation of the mouse homologue mCCL20 gene message by IL-17
The evolutionary conservation of IL-17-induced CCL20 expression was tested by analyzing the expression of its mouse homologue, mCCL20, by mouse primary tracheal epithelial cells. Various concentrations of IL-17 were added to these cultures, and RNA was harvested 24 h later for real-time RT-PCR quantification as described in Materials and Methods. The baseline mCCL20 expression in mouse TBE cells was lower than human CCL20 expression, but IL-17 was still able to significantly increase mCCL20 message in primary mouse TBE cells (Fig. 4). Peak stimulation occurred at 100 ng/ml dose, with no further increase at 200 ng/ml level. This result further supports the potency of IL-17 in enhancing CCL20 expression by various airway epithelial cells.
IL-17-stimulated CCL20 secretion bidirectionally
IL-17 also stimulated CCL20 peptide secretion in primary human TBE cultures. As shown in Fig. 5, both apical and basolateral secretions of CCL20 peptide were significantly increased in these ALI cultures after IL-17 treatment. Because the volumes of the apical and basolateral secretions are considerably different, it is more accurate to make a comparison of secretion per Transwell chamber. On the apical side of the culture chamber, IL-17 significantly increased CCL20 secretion from a baseline of 0.56 ± 0.02 to 1.82 ± 0.08 ng/chamber 24 h after treatment. On the basolateral side of the secretion, IL-17 increased CCL20 secretion from a baseline of 6.10 ± 0.22 to 23.02 ± 1.11 ng/chamber. These results suggest that IL-17 also stimulates the production of CCL20 at the peptide level. Although IL-17 stimulates CCL20 secretion ∼3.5-fold on both sides, >90% of the total CCL20 secretion was toward the basal side of the culture chamber.
Differential effects of apical and basolateral IL-17 treatments on CCL20 expression
To gain an insight into the effectiveness of IL-17 treatment on the induction of CCL20 expression, the polarized ALI cultures were treated with IL-17 on either the apical and/or basal side of the culture. As shown in Fig. 6,A, there was no stimulation of CCL20 in cultures that were treated with IL-17 on the apical side only. In contrast, the treatment from the basal side was as effective as the treatment from both the apical and basal sides of the culture. To further demonstrate the polarity, anti-IL-17RA Ab was used to stain the receptor distribution in these ALI primary TBE cultures. Using the confocal microscope, a polarized expression of IL-17RA (Alexa Fluor 488, green stain) was mainly seen on the basolateral side of the culture (Fig. 6 B). These results are consistent with the treatment study to further suggest the polarity of IL-17RA expression in these ALI primary TBE cultures.
Inhibition of IL-17-stimulated CCL20 expression by inhibitors of NF-κB and MEK1/2 but not by inhibitors of JAK, PI3K, p38, and JNK pathways
To elucidate the possible signaling pathways involved in IL-17-enhanced CCL20 expression, inhibitors of various signaling pathways were used (Fig. 7). Among these inhibitors, we found that helenalin, an inhibitor for the DNA-binding activity of NF-κB p65 subunit (28), was the most potent inhibitor to suppress the IL-17-induced CCL20 expression. As shown in Fig. 7,C, helenalin inhibition of IL-17-mediated CCL20 expression was dose-dependent. In contrast, both the JAK universal inhibitor (JAK inhibitor I) (Fig. 7,A) and PI3K inhibitor (LY29402) (Fig. 7,B) had no suppressive effect on IL-17-stimulated CCL20 expression. Because it has been reported that IL-17 may activate the MAPK pathways (29, 30, 31), inhibitors of these pathways were used to see whether any of these pathways were involved. As shown in Fig. 7, U0126, an inhibitor of MEK1/2, was very effectively to suppress IL-17-induced CCL20 expression and a dose-dependent suppression was observed (Fig. 7,D). In contrast to this finding, JNK inhibitor II had a significant super-enhancement of IL-17-stimulated CCL20 expression at low doses (Fig. 7,E). At the highest dose (50 μM), the super-enhanced phenomenon was not noted, presumably due to nonspecific nature of the inhibitor. For p38 pathway, the specific inhibitor SB203580 had no effect at any dose (Fig. 7 F).
Induced p65 nuclear translocation by IL-17
To confirm NF-κB activation, induced p65 nuclear translocation was conducted. As shown in the upper panel of Fig. 8, before IL-17 treatment, a majority of anti-p65 staining (green, Alexa Fluor 488) was in the cytoplasm of cultured TBE cells. There was no green staining in the nucleus, as shown by the DAPI-specific blue fluorescent stain. Thirty minutes after IL-17 treatment, nuclear translocation of p65 was clearly seen in many of these TBE cells, although a small fraction of these cells still retained p65 in their cytoplasm (Fig. 8, middle panel). As a comparison, p65 nuclear translocation was more extensive in these TBE cells after IL-1β treatment (Fig. 8, lower panel). These results are consistent with the NF-κB inhibitor study, further suggesting the significant role of IL-17-mediated NF-κB activation in the regulation of CCL20 expression.
Previously we observed that IL-17 could stimulate CCL20 expression through Affymetrix gene chip-based microarray analysis (19). The current study further extends the microarray data through a quantitative real-time RT-PCR approach and also provides three additional findings. First, through a comparison with a panel of cytokines, we have shown that IL-17 is as potent as TNF-α, IL-1α, and IL-1β in stimulating CCL20 expression. This finding together with the previous study of IL-17-dependent hBD-2 expression (19) suggests an important role of IL-17 in the regulation of airway innate immunity. Second, we have shown that IL-17-dependent CCL20 expression is independent from JAK-mediated signaling but sensitive to inhibitors of MEK1 pathway and NF-κB-mediated transcriptional regulation. The JAK-independent phenomenon is in contrast to the JAK-dependent IL-17-mediated hBD-2 expression (19). Despite a coordinated induction of CCL20 and hBD-2 expression by IL-17, the regulation of these genes is different. In addition to these divergent signaling pathways, the third finding has shown a polarity of IL-17 effect and the distribution of these innate molecules in well-differentiated epithelium. For both CCL20 and hBD-2, IL-17 treatment is effective only from the basal side of the epithelial layer, whereas the stimulated secretions for CCL20 and hBD-2 are different with a preferential secretion toward basal and apical sides of the culture, respectively.
We have demonstrated that IL-17 is as potent as TNF-α, IL-1α, and IL-1β in stimulating CCL20 expression (32). Inductions of CCL20 by IL-1α, IL-1β, and TNF-α treatment are consistent with previous reports (10, 33, 34). However, we could not reproduce the stimulatory effect of Th2 cytokines, as reported recently by Gordon and colleagues (17). The nature of the discrepancy between this study and the other is difficult to assess. One possible difference might be related to the culture conditions used in these studies. The primary TBE cells used in this study were cultured under ALI conditions and treated continuously with IL-17 until the cells reached to a well-established mucociliary stage with evidences of cilia beating, mucin secretion (measured by ELISA), and transepithelial resistance. Gordon and colleagues (17) used immersed cultures, which may not differentiate as well. The response of TBE cells to cytokines may depend upon their state of differentiation.
CCL20 is constitutively expressed in a variety of normal human mucosa-associated tissues, especially in the mucosal epithelial cells, including the airways surface epithelia cells (35, 36, 37). Although the up-regulation of CCL20 by IL-17 has been reported in synoviocytes and cultured primary keratinocytes (32, 38, 39), the influence of IL-17 on CCL20 in airway epithelial cells has not been reported before. IL-17 is an important cytokine that has gained more recognition for its role in various airway diseases. Initially, IL-17 was found to play a major role in antimicrobial infections (20, 40). Lately, the level of IL-17 has been found significantly elevated in BALF of various chronic lung diseases (41, 42), including allergy-induced asthma (23). We have previously shown that IL-17 is one of the most potent cytokines to stimulate the expression of mucin genes, MUC5AC and MUC5B, and hBD-2 in primary human TBE cells (18, 19). Both CCL20 and hBD-2 have a potent antimicrobial activity and chemotactic activity to CCR6-positive dendritic and T cells (4). Although these two peptides have no sequence homology and they belong to two different gene families, they share many similarities in terms of their antimicrobial and chemotactic activities, binding to CCR6 receptor, regulation by IL-1β and TNF-α, and now, the coordinated regulation by IL-17.
Several previous reports have shown that IL-17 may augment or enhance certain gene expression via stabilizing the mRNAs. For example, IL-17 was able to stabilize the IκB-ζ mRNA in NIH 3T3 cells (27). It has also been reported previously that LPS is able to prolong CCL20 mRNA turnover in neutrophils in the presence of fMLP or IFN-γ (11). Thus, it is possible that a part of IL-17-stmulated CCL20 expression is through the enhancement of mRNA stability. However, results from the actinomycin D experiments do not support this possibility. Instead, we have found that CCL20 message has a relative long half-life, regardless of IL-17 treatment, compared with the β-actin message in TBE cells. This stability is in contrast to the IκB-α message, which is well known for its instability in cells (Fig. 3). Therefore, it is unlikely that a regulatory step at the posttranscriptional level is involved in IL-17-induced CCL20 expression.
Consistent with the above notion of possible transcriptional regulation, we found that helenalin, an inhibitor for the DNA-binding activity of NF-κB p65 subunit (28), could dramatically suppress IL-17-stimulated CCL20 expression in a dose-dependent fashion. A similar sensitivity to this inhibitor was seen in IL-17-mediated hBD-2 expression (19). Although helenalin induces apoptosis (10–50 μM) in Jurkat T cells (43), there were no effects on cell viability or on total RNA levels at concentrations of helenalin as high as 50 μM, suggesting that the inhibitory effect observed in primary TBE cells was not due to toxicity (data not shown). Another study had used a similar level of helenalin to inhibit NF-κB-based transcriptional regulation in pulmonary epithelial cells (9). Although there might be nonspecific effect at the highest dose of helenalin, we have further demonstrated IL-17 induced-NF-κB activation by enhancing the nuclear translocation of p65 from the cytoplasm (Fig. 8). IL-17 has been shown to activate NF-κB in several other cell types (30, 44, 45). This study and the ones previous suggest that IL-17 is a potential inducer for NF-κB-dependent transcriptional events, a common property shared by various proinflammatory cytokines, including TNF-α and IL-1β.
To elucidate the upstream signaling pathways involved in CCL20 induction, various inhibitors were used in this study. To our surprise, we found that CCL20 induction was not blocked by JAK inhibitor I, which is a pan inhibitor for various receptor-associated JAK gene family members (JAK-1, -2, -3, and Tyk-2). This result is in contrast to the regulation of hBD-2, which is sensitive to JAK inhibitor I (19). Although IL-17 has a low affinity with its receptor (IL-17RA), it has been shown in human U937 monocytic leukemia cells that various JAK isotypes (JAK-1, -2, -3, and Tyk-2) could interact with IL-17RA upon the binding of its ligand (46). The insensitivity to JAK inhibitor 1 in this study suggests that an alternative signaling pathway, other than the traditional receptor/JAK activation system is involved in IL-17-induced CCL20 expression. To elucidate this alternative signaling pathway is beyond the scope of this communication.
In addition to JAK activation by the receptor-ligand interaction, PI3K and MAPK pathways have been shown to be involved in IL-17-stimulated gene expression in various cell types (29, 31, 47). In this study, we found that inhibitors of PI3K and two of MAPK pathways, JNK and p38, could not attenuate IL-17-induced CCL20 expression. There was a superinduction of the expression by the JNK inhibitor. The nature of this superinduction is not clear to date. However, the MEK1/2 inhibitor U0126 showed a dose-dependent inhibitory effect to CCL20 induction, with a significant inhibition observed as low as 2 μM. We have found no toxicity to cells at these doses (2–50 μM). Others have used a similar range of doses for their MEK1/2 inhibitor studies on airway epithelial cells (48, 49). Higher concentrations (up to 100 μM) of U0126 have proven to keep a selective effect on ERK, JNK, and p38 phosphorylation in human monocytes (50), whereas <20% of JNK or p38 activities were inhibited by U0126 at lower doses. Therefore, it is less likely that the effect of U0126 is due to its nonspecific nature on pathways other than MEK1/2. Interestingly, U0126 was shown to partially inhibit NF-κB-binding activity and the NF-κB-dependent transcription as well as the degradation of IκB in mouse proximal tubular cells (51). Recent findings in melanoma cells also suggest that NF-κB-induced kinase (NIK) was found to regulate NF-κB activation through a novel NIK-MEK-ERK-NF-κB signaling pathway in addition to the classical NIK-IKK-IκB-NF-κB pathway (52). These results suggested a possible cross-talk between the MEK-ERK pathway and the IκB-NF-κB activation existed in IL-17-treated TBE cells.
In addition to the molecular mechanism of induction, we also demonstrated a polarity effect of IL-17 treatment and the bidirectional secretion of CCL20 by TBE cells. We have observed that the treatment of IL-17 from the basolateral side of the polarized TBE cultures was the most effective way to stimulate CCL20 expression. Treatment of IL-17 from the apical side of the culture produced no stimulation. A similar effect was also seen for IL-17-enhanced hBD-2 expression (data not shown). Thus, it is possible that the distribution of the IL-17 receptor, especially IL-17RA form, is largely located at the basal layer of the polarized TBE culture. This notion is further confirmed by a confocal fluorescence microscope study, which showed a preferential immunofluorescent staining on the basal side of the culture with an Ab specific to the native form of IL-17RA (Fig. 6 B). We have shown previously that this Ab can block IL-17-enhanced hBD-2 expression (19). Consistent with this finding, Kolls and colleagues (53) have shown recently that IL-17RA is indeed expressed on the basolateral side of well-differentiated trachea epithelial cells in vitro.
We also observed a bidirectional secretion of CCL20 in both steady and IL-17-stimulated conditions. Based on ELISA data, we found that polarized TBE cultures secreted 0.56 ± 0.02 ng per day (at a confluent culture density of 2 million cells/chamber) to the apical side of the culture, whereas 6.1 ± 0.22 ng was secreted to the basolateral side of the underneath culture medium. These secretions toward the apical and basal compartments were coordinately elevated to 1.82 ± 0.08 and 23.02 ± 1.11 ng levels, respectively, in cultures after IL-17 treatment. Although there was a similar 3- to 4-fold stimulation of the secretion in both directions by IL-17, the apical secretion accounted for <10% of the total amount of CCL20 secretion. These results support a bidirectional secretion of CCL20, with a preferential secretion toward the basolateral side by airway epithelial cells. Furthermore, IL-17 has stimulatory effects with no apparent influence on the property of the secretion. These results are opposite to IL-17-induced hBD-2 secretion, which is preferential toward the apical side of the culture (19). Because CCL20 and hBD-2 shared similar functions, the differential secretion but coordinate stimulation by IL-17 may suggest a complementary activity in exerting the innate nature of IL-17 in host defense.
IL-17, mainly secreted by activated T cells (54, 55), is prominently elevated in the airway lumen after microbial infections (40, 56) and has a proinflammatory role in mediating neutrophil migration (57) and the production of IL-6 and IL-8 (58, 59). Because IL-17 is a very potent cytokine in the stimulation of CCL20 and hBD-2, and IL-17 elevation in airways has been related to the microbial infection (22), the following innate/adaptive immune response model of IL-17 in respiratory airways is proposed. We postulate that during the early stage of bacterial infection in the airways, microbial products such as LPS bind to pattern recognition receptors such as the TLR4 on epithelial cells and directly or indirectly stimulate a low level induction of CCL20 and hBD-2 (9, 12, 60). The innate nature of this response may provide an initial antimicrobial defense. After the acquired immune response has time to develop, certain types of T cells are chemoattracted to the site of infection in the subepithelial compartment to locally release IL-17. The locally secreted IL-17 further enhances the antimicrobial activity by binding to the IL-17 receptor underneath epithelial cells via an NF-κB-dependent gene expression mechanism for the expression of hBD-2 and CCL20 genes. Interestingly, present and previous studies (19) have shown that IL-17 can exert signaling pathways through JAK-dependent and -independent activation of NF-κB for stimulating hBD-2 and CCL20 expression, respectively, in the same airway epithelial culture. Noteworthy is that the secretion of hBD-2 is mainly apical, whereas CCL20 secretion is basolateral. Such a scenario in the coordination and differential secretions exerted by a single cytokine IL-17 may have a very significant implication in protecting airways against the microbial infection. For hBD-2, a preferentially apical secretion is consistent with the antimicrobial nature of this molecule. A preferential secretion of CCL20 to the basolateral side of airways would support the chemotactic function of this cytokine in recruiting other leukocyte types, including memory T cells to the infected sites of airways to ensure the clearance of invaders. The cross-talk between IL-17-induced gene expression and the recruitment of various inflammatory cells might be instrumental to direct the transition from an innate immunity to an adaptive immune response.
In summary, the present study demonstrates that IL-17 directly stimulates CCL20 gene expression in primary airway epithelial cells. This effect mainly occurs through the basolateral treatment of cells with IL-17. It appears that there is a coordinate expression and secretion between hBD-2 and CCL20 in airway epithelia after IL-17 treatment. Further studies on the molecular basis of the coordinated activation of NF-κB by IL-17-induced JAK-dependent and -independent signaling pathways are needed. Our results with IL-17 widen the spectrum of known cytokines that can regulate CCL20 expression and offer preliminary evidence for a novel mechanism in coordinating both innate and adaptive immune responses.
We thank Yu Hua Zhao for suggestions on performing the ELISA for CCL20 and the superior cell culture work in providing primary human TBE cell cultures used in the study. Dr. Suzette Smiley-Jewell is thanked for editing of the manuscript.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by Grants HL35635, HL077315, HL077902, ES00628, and AI 50496 from the National Institutes of Health. This work was also supported by the National Institutes of Health T32 Training Grant HL07013.
Abbreviations used in this paper: hBD, human β-defensin; TBE, tracheobronchial epithelial; ALI, air-liquid interface; BALF, bronchoalveolar lavage fluid; NIK, NF-κB-induced kinase.