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Airway Epithelial Cells Release MIP-3/CCL20 in Response to Cytokines and Ambient Particulate Matter

Division of Pulmonary and Critical Care Medicine, Department of Medicine, and Department of Environmental Medicine, Nelson Institute of Environmental Medicine, New York University School of Medicine, New York, New York

Address correspondence to: Joan Reibman, M.D., New York University School of Medicine, Division of Pulmonary and Critical Care Medicine, 550 1st Avenue, Room NB7N24, New York, NY 10016. E-mail: reibmj01{at}gcrc.med.nyu.edu

	Abstract

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The initiation and maintenance of airway immune responses in Th2 type allergic diseases such as asthma are dependent on the specific activation of local airway dendritic cells (DCs). The cytokine microenvironment, produced by local cells, influences the recruitment of specific subsets of immature DCs and their subsequent maturation. In the airway, DCs reside in close proximity to airway epithelial cells (AECs). We examined the ability of primary culture human bronchial epithelial cells (HBECs) to synthesize and secrete the recently described CC-chemokine, MIP-3/CCL20. MIP-3/CCL20 is the unique chemokine ligand for CCR6, a receptor with a restricted distribution. MIP-3/CCL20 induces selective migration of DCs because CCR6 is expressed on some immature DCs but not on CD14⁺ DC precursors or mature DCs. HBECs were stimulated with pro-inflammatory cytokines tumor necrosis factor- and interleukin (IL)-1ß or, because of their critical role in allergic diseases, IL-4 and IL-13. Cells were also exposed to small size-fractions of ambient particulate matter. Each of these stimuli induced MIP-3/CCL20 gene and protein expression. Moreover, these agents upregulated mitogen-activated protein kinase pathways in HBECs. Inhibition of the ERK1/2 pathway or p38 reduced cytokine-induced MIP-3/CCL20 expression. These data suggest a mechanism by which AEC may facilitate recruitment of DC subsets to the airway.

Abbreviations: airway epithelial cell, AEC • analysis of variance, ANOVA • bronchial epithelial cell growth medium, BEGM • dendritic cell, DC • enzyme-linked immunoabsorbent assay, ELISA • granulocyte-macrophage colony stimulating factor, GM-CSF • human bronchial epithelial cell, HBEC • interleukin, IL • lipopolysaccharide, LPS • mitogen-activated protein kinase, MAPK • macrophage inhibitory protein, MIP • particulate matter, PM • polyvinylidene difluoride, PVDF • reverse transcription polymerase chain reaction, RT-PCR • tumor necrosis factor-, TNF- • ultrafine, UF

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The initiation and maintenance of airway immune responses in Th2-type allergic diseases (e.g., asthma) are dependent on the specific activation of local airway dendritic cells (DCs) (1, 2). DCs, which are derived from bone marrow, migrate at an immature stage to epithelial cell surfaces. At this immature stage, DCs are capable of antigen capture, which can occur via receptor-mediated endocytosis, macropinocytosis, or phagocytosis (1, 2). Upon exposure and uptake and processing of specific antigens at the epithelial cell surface, DCs begin the process of maturation and migration away from the epithelium to regional lymph nodes. Maturing DCs, now incapable of antigen capture, present antigen in the context of MHC Class II peptide complexes and, in the presence of costimulatory molecules and in the appropriate cytokine microenvironment, induce a polarized T-cell response (1–4). DCs may present antigen in such a way as to induce a Th1 or Th2 response (4). The polarization of DCs is believed to be determined by the cytokine microenvironment at the site of DC maturation. In the airway, this microenvironment is induced in part by the airway epithelial cell (AEC). The recruitment of immature DCs to the airway and the site of antigen exposure is tightly regulated and is dependent on the release of select chemokines and the expression of specific chemokine receptors by DCs (5). Chemokines, which are small-molecular-mass proteins that regulate leukocyte migration via the activation of seven transmembrane-spanning G protein-coupled receptors, have been defined into subfamilies depending on their sequence homology and the position of the first two cystein residues (6–8). MIP-3/CCL20 is a CC chemokine recently described through bioinformatics (9–11). MIP-3/CCL20 is a unique functional ligand for the chemokine receptor CCR-6. This receptor is selectively expressed on immature DCs, such as Langerhans cell precursors, a subpopulation of DCs that reside at mucosal surfaces (12). It is also expressed on distinct subpopulations of Ag-activated CD4⁺ T lymphocytes and B cells (13, 14). Upon maturation, DCs downregulate expression of CCR6 and lose their ability to respond to MIP-3/CCL20 while they upregulate CCR7 and gain the ability to respond to MIP-3ß. MIP-3/CCL20 has been demonstrated in tonsillar crypts, inflamed intestinal epithelial cells, and keratinocytes (15–18). Thus, it is possible that MIP-3/CCL20 functions as an important chemokine for the recruitment of a distinct population of CCR6-expressing immature DCs to the airway for subsequent antigen presentation.

Rapid recruitment of DCs into the bronchial mucosa has been well documented in animal studies and in human subjects in response to allergen challenge (19, 20). The recruitment of these DCs in response to bacterial-induced inflammation is dependent upon the interaction between CCR1 and CCR5 with RANTES (21). The recruitment of DCs in response to challenge with virus or soluble recall Ag requires an additional mechanism that has not been described (21). The possibility exists that this additional signal may involve the interaction between CCR6 and MIP-3/CCL20. The importance of CCR6 in allergic pulmonary inflammation has recently been demonstrated using a cockroach antigen model with CCR6-/- mice (22). These mice demonstrate a diminished allergic response with reduced peribronchial eosinophil accumulation and IgE production.

The local cytokine environment that modifies the recruitment, maturation, and polarization of airway DCs is produced in part by AECs. AECs are adjacent to and are in close association with dendritic cells (23). AECs are one of the first targets of ambient stimuli. These stimuli may result in AEC release of select cytokines/chemokines that influence the recruitment and subsequent activation of local DC populations. We hypothesized that AECs would up-regulate MIP-3/CCL20 in response to ambient stimuli and cytokines. We evaluated the effects of proinflammatory cytokines tumor necrosis factor (TNF)- and interleukin (IL)-1ß, and, because of their elevated levels and importance in asthma, IL-4 and IL-13 on this response. Because of the interest in ambient pollutants as immune response modifiers, we also investigated the effect of ambient particulate matter (PM) on MIP-3/CCL20 production, using small size-fractionated ambient PM. Furthermore, we investigated whether MIP-3/CCL20 was induced by mitogen-activated protein kinases (MAPK). Our data suggest that MIP-3/CCL20 release by AEC was induced by a broad spectrum of cytokines and ambient agents. Moreover, these effects involved rapid activation of MAPK pathways.

	Materials and Methods

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Reagents
Basal cell culture medium was obtained from Clonetics (San Diego, CA) and was supplemented with the following compounds to generate culture medium (bronchial epithelial cell growth medium [BEGM]): hEGF (0.5 ng/ml), hydrocortisone (0.5 µg/ml), insulin, (5 µg/ml), transferrin (10 µg/ml), epinephrine (0.5 µg/ml), triiodothyronine (6.5 ng/ml), gentamicin (50 µg/ml), amphotericin-B (50 ng/ml), bovine pituitary extract (13 µg/ml), and retinoic acid (0.1 ng/ml). Human recombinant TNF-, IL-1ß, IL-4, and IL-13 were obtained from R&D systems (Minneapolis, MN). Antibodies directed against phosphorylated and nonphosphorylated forms of ERK and p38 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and Cell Signal Technology (Beverly, MA). PD98059 and SB203580 were obtained from Calbiochem (La Jolla, CA). ECL Plus kits were obtained from Amersham Pharmacia (Buckinghamshire, UK).

Cell Culture
Normal human bronchial epithelial cells (HBECs) were derived from bronchial brush biopsies (New York University Institutional Review Board-approved protocol) performed with a disposable brush (Model BC-15C; Olympus, Melville, NY). Cells were collected into BEGM and were plated in uncoated T25 tissue culture flasks and incubated (37°C, 5% CO₂) for 7–10 d, during which time the cells were fed every 3 d. When cells reached 70% confluence, they were passaged into appropriate tissue culture plates. Alternatively, cells were obtained from BioWhittaker (Walkersville, MD). All experiments were performed at Passage 3. Hydrocortisone, retinoic acid, and epinephrine were removed from the medium 24 h before each experiment. When indicated, an SV-40-adenovirus–transformed bronchial epithelial cell line (BEAS-2B; American Type Culture Collection, Manassas, VA) was used. Cells were cultured in T75 flasks (37°C, 5% CO₂) and treated in a similar manner. Cells were used between Passages 22–35.

PM
Size-fractionated samples of ambient PM were collected from New York City air using four stages of a cascade impactor (micro-orifice uniform deposition impactor; MOUDI, MSP Corp., St. Paul, MN) located in downtown Manhattan, New York City (8th floor of a building located on 26th St. and 1st Ave). The MOUDI is a cascade impactor that uses micro-orifice nozzles to extend the cut sizes of the lower stages without going to low pressures or creating excessive pressure drops across the impactor stages. A sealed transport cover for impaction plates and filter holders was used to allow transport without contamination. A pre-impactor screen prevented insects and rain or snow droplets from entering the impactor. The first stage removes nuisance particles and was not used for biologic assays. The particles collected on filters and the after-filter particles do not strictly correspond to standard Environmental Protection Agency definitions of PM₁₀ and PM_2.5. For the purposes of the study, we defined sizes to closely approximate standard definitions as follows: ultrafine (UF)/fine <0.18 µm; fine 0.18–1.0 µm; intermediate 1–3.2 µm; coarse >3.2 µm.

Samples were collected (14 d) on inert filters (Nucleopore or Teflon filters) used as impactor substrates (endotoxin-free). Impactor substrates and after-filters were weighed before and after sampling on a Cahn electrobalance (1 µg sensitivity) (Cahn Instruments, Madison, WI)), and PM was removed by ultrasonification (20 min) into sterile medium in a sterile 50-ml conical polypropylene tube followed by ultrasonification (10 sec x 3) with a Virsonic 50 ultrasonicator (Virtis, Gardiner, NY). Samples were suspended in medium used for the growth of HBECs. HBECs were exposed to a dose of 100 µg/ml (11 µg/cm²), and cultures were examined for changes in morphology and adhesion using an inverted microscope. Gross alterations were not detected in cellular morphology or adhesion in UF/fine PM-exposed cells compared with control cells. Toxicity was measured by trypan blue exclusion, and cells were >90% viable.

Carbon particles were generated by passing a small quantity of acetylene in argon into a high-temperature silicon carbide furnace that was maintained at 1098°C. The acetylene underwent a thermal decomposition and produced carbon black particles. These carbon particles were collected using a cascade impactor.

Reverse Transcription Polymerase Chain Reaction
Total RNA was isolated using TRIzol reagent (Gibco BRL, Grand Island, NY). Reverse transcription polymerase chain reaction (RT-PCR) was performed using SuperScript One-step RT-PCR (GibcoBRL, Rockville, MD) according to the manufacturer's protocol. The reaction was performed in a reaction mixture containing RT/PLATINUM Taq with a Perkin Elmer DNA thermal cycler (Perkin Elmer, Shelton, CT). cDNA synthesis was performed with one cycle at 55°C for 30 min, followed by 94°C for 2 min. mRNA amplification was performed by 30 cycles of PCR with the following conditions: 94°C for 1 min, 61°C for 2 min, and 72°C for 3 min. Final extension was performed at 72°C for 10 min. PCR products were visualized on 1.5% agarose gels containing 0.5 µg/ml ethidium bromide. The primers for MIP-3/CCL20 were 5'-CACAGACCGTATTCTTCATCCTAAATTTATTG-3' (forward primer) and 5'-CCCCAGCAAGGTTCTTTCTGTTCTTGGGCTATTGCC-3' (reverse primer).

Enzyme-Linked Immunosorbent Assay
Cells were grown to near confluence at Passage 3 and stimulated with the specified agents (18 h, 37°C, 5% CO₂). Supernatants were collected, and the concentration of MIP-3/CCL20 was determined by a commercial enzyme-linked immunoabsorbent assay (ELISA) (Endogen; Cambridge, MA or R&D Systems, Minneapolis, MN) with a sensitivity 0.1 pg/ml. Optical density was measured at 450 nm according to the manufacturer's instructions.

Immunoblotting with Phosphospecific Antibody Probes
Cells were incubated in basal medium (4 h) before stimulation with defined agents. Cell lysates were prepared with lysis buffer (20 mM Tris-HCL [pH 7.4], 150 mM NaCl, 0.5% Triton X 100, 1% sodium deoxycholate, 0.5 M PMSF, 2 mM Na₃VO₄, 50 mM NaF, 1 mM EGTA, and antiproteases). Equal amounts of protein were electrophoresed in 10% SDS-Tris glycine gels and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were probed with anti-phospho ERK1/2 (1:200), anti-p38 (1:1000), or anti-ERK2 (1:200). Membranes were subsequently incubated with the appropriate horseradish peroxidase-conjugated secondary antibody. Bound antibodies were visualized using the ECL Western blot detection system (Amersham, Piscataway, NJ) according to the manufacturer's instruction.

Statistical Analysis
Results are expressed as the means of three of more determinations ± SEM as indicated. Data were examined by repeated measures analysis of variance (ANOVA). Post hoc comparisons were considered statistically significant at P < 0.05. Immunoblots are representative of individual experiments that were confirmed by three or more separate studies.

	Results

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Expression of MIP3/CCL20 mRNA
To assess whether primary culture HBECs had the capacity to secrete MIP3/CCL20, we first investigated the expression of MIP3/CCL20 mRNA in response to pro-inflammatory stimuli. HBECs were stimulated with TNF- or IL-1ß (5 and 10 ng/ml, respectively; 6 h, 37°C). Total RNA was isolated using TRIzol reagent, and mRNA for MIP3/CCL20 was detected using RT-PCR. MIP3/CCL20 mRNA was minimally detected in resting HBECs (Figure 1) . TNF- and IL-1ß induced marked mRNA expression at 6 h. Levels of IL-4 and IL-13 are abundant in the diseased airway (e.g., in asthma) and have been implicated as critical cytokines in asthma (24). We therefore tested whether these Th2 cytokines increased MIP3/CCL20 mRNA expression in HBECs. IL-4 (10 ng/ml) and IL-13 (10 ng/ml) induced an increase in mRNA expression at the same time point as TNF- and IL-1ß (Figure 1). Because of the increasing interest in diesel components and PM in immune diseases such as asthma, we tested whether UF/fine ambient PM, the smallest size fraction of ambient PM that we collected, was capable of increasing the expression of MIP3/CCL20 mRNA. Exposure of HBECs to UF/fine PM (100 µg/ml) induced an increase in MIP3/CCL20 mRNA (Figure 1). These data suggest that MIP3/CCL20 expression could be upregulated by a broad spectrum of pro-inflammatory and immunomodulatory cytokines and by ambient particles.

View larger version (31K): [in this window] [in a new window]   Figure 1. Expression of MIP-3/CCL20 mRNA by HBECs. HBECs were stimulated with defined agents (6 h, 37°C), and total RNA was isolated using TRIzol reagent from resting cells (Lane 1), or cells were stimulated with TNF- (5 µg/ml, Lane 2), IL-1ß (10 µg, Lane 3), IL-4 (10 µg/ml, Lane 4), or UF/fine PM (100 µg/ml, Lane 5). RT-PCR was performed using primers for MIP-3/CCL20 and GAPDH as a control as described in MATERIALS AND METHODS. Numbers below the gel represent densitometry ratio of MIP-3/CCL20/GAPDH.

Cytokine-Induced Secretion of MIP3/CCL20 by HBECs

View larger version (19K): [in this window] [in a new window]   Figure 2. Release of MIP-3/CCL20 by HBECs. HBECs (Passage 3) were stimulated (18 h, 37°C) in the absence or presence of increasing concentrations of (A) TNF- or IL-1ß or (B) IL-4 or IL-13. Medium was removed and assayed for MIP-3/CCL20 release by ELISA. Data are presented as the mean ± SEM (n = 3; *P < 0.05 [ANOVA], stimulated versus unstimulated).

The chemokines MIP-1/CCL3 and RANTES/CCL5 (regulated upon activation, normal T-cell expressed and secreted) are also capable of recruiting immature DCs (5). We tested whether HBECs released MIP-1/CCL3 and RANTES/CCL5 in response to concentrations of TNF- (5 ng/ml), IL-1ß (10 ng/ml), IL-4 (10 ng/ml), or IL-13 (10 ng/ml) that induced MIP-3/CCL20. HBECs were stimulated (18 h, 37°C) with each agent, and the supernatants were tested for these chemokines using a commercially available ELISA. We were unable to detect the release of MIP-1/CCL3 or RANTES/CCL5 in response to these concentrations of cytokine in our system (data not shown; n = 3, P = ns).

Secretion of MIP-3/CCL20 in Response to Ambient PM
The ability of ambient particulate matter to modify immune diseases such as asthma is under increasing scrutiny. We have previously demonstrated that small particles of ambient PM derived from New York City air are capable of upregulating GM-CSF (25). We investigated whether these particles were capable of increasing the release of MIP-3/CCL20 by HBECs. Size-fractionated samples of ambient PM were collected over 2-week periods from New York City air using four stages of a cascade micro-orifice uniform deposition impactor located in downtown Manhattan. HBECs were exposed to the smallest size fractions of ambient PM consisting of UF/fine, fine, or intermediate particles. We used a concentration of 100 µg/ml (11 µg/cm²) because we and others have previously demonstrated that this concentration induces the upregulation of cytokines such as GM-CSF (26). The coarse fraction was not tested because of limited recovery of particles. Release of MIP-3/CCL20 in cell supernatants was determined by ELISA. Resting HBECs released a minimal amount of MIP-3/CCL20 in the supernatant (Figure 3) . MIP-3/CCL20 release increased in response to small size fractions of ambient PM (repeated measures ANOVA, P < 0.05). A significant increase in MIP-3/CCL20 was induced by fine and medium particles, with a trend toward significance (P < 0.073) with UF/fine ambient PM. To confirm that MIP-3/CCL20 release was caused by ambient PM and not a general particle effect, similar studies were performed using generated size-fractionated UF/fine carbon particles. These particles failed to induce MIP-3/CCL20 release. To confirm that the responses identified were not due to possible filter contamination, we tested the effect of medium that had been treated in the same manner as that into which ambient particles were collected. Filter medium failed to upregulate MIP-3/CCL20 (Figure 3). In additional experiments, lipopolysaccharide (LPS) (0.01–1.0 µg/ml) failed to upregulate MIP-3/CCL20 (data not shown). We were unable to detect the release of MIP-1/CCL3 or RANTES/CCL5 in response to these size fractions of PM in our system (data not shown; n = 3, P = ns).

View larger version (15K): [in this window] [in a new window]   Figure 3. Release of MIP-3/CCL20 by HBECs in response to ambient PM. HBECs were stimulated (18 h, 37°C) in the absence or presence of size-fractionated ambient PM (100 µg/ml), carbon particles, or medium derived from sham filter. Data are presented as the mean ± SEM (n = 3–4; *P < 0.05 [repeated measures ANOVA], stimulated versus unstimulated).

To demonstrate whether the activation of ERK1/2 was associated with MIP-3/CCL20, we first asked whether we could identify the upregulation of ERK1/2 in response to all four cytokines that induced MIP-3/CCL20 mRNA and protein. HBECs were treated (15 and 60 min) with TNF-, IL-1ß, IL-4, or IL-13 (5 ng/ml). Western immunoblots were performed on cell lysates using antibodies that recognized the phosphorylated (and thus activated) form of ERK1/2. Resting cells displayed minimal expression of phosphorylated ERK1/2 (p-ERK1/2) (Figure 4A) . A rapid (15-min) increase in p-ERK1/2 was detected in response to TNF-, IL-1ß, IL-4, and IL-13. The response was sustained at 60 min with all four stimuli. These effects were not due to changes in total protein as confirmed by normalizing expression of p-ERK1/2 to pan ERK2.

View larger version (64K): [in this window] [in a new window]   Figure 4. Activation of MAPK in HBECs. Lysates were prepared from resting HBECs (Lane 1), or HBECs were stimulated with TNF- (5 ng/ml, Lanes 2 and 6), IL-1ß (5 ng/ml, Lanes 3 and 7), IL-4 (5 ng/ml, Lanes 4 and 8), or IL-13 (5 ng/ml, Lanes 5 and 9) for 15 or 60 min, respectively. After SDS-PAGE and transfer to PDVF membranes, immunoblotting was performed with (A) phosphospecific (p-ERK 1/2) or control (pan-ERK2) probes, or (B) immunoblotting was performed with phosphospecific p38 (p-p38) or control pan-ERK2 probes. Numbers at the bottom of the lanes are the ratio of the densitometry of phosphorylated over control antibody to correct for variations in protein loading.

To test the importance of the activation of ERK1/2 on the cytokine-induced release of MIP-3/CCL20, we used the cell permeant molecule PD98059 (4 µM), a selective inhibitor of the upstream MAPK extracellular-regulated kinases (MEK1/2), which phosphorylate and activate ERK1/2. To confirm that the activation of p-38 was associated with MIP-3/CCL20 release, cells were exposed to the defined cytokines in the presence of SB203580 (0.1 µM), an inhibitor of p38 MAPK. BEAS-2B displayed little constitutive release of MIP-3CCL20 (Figure 5) . However, BEAS-2B cells were capable of abundant release of MIP-3/CCL20 in response to all four cytokines. PD98059 inhibited MIP-3/CCL20 release induced by BEAS-2B cells in response to IL-1ß, IL-4, and IL-13 with 75.0 ± 7.0, 52.6 ± 18.8, and 75.5 ± 0.5% inhibition in response to PD98059, respectively. In addition, exposing cells to SB203580 resulted in significant inhibition of IL-1ß–, IL-4–, and IL-13–stimulated MIP-3/CCL20 release with 79.0 ± 3.7, 70.7 ± 11.4, and 83.5 ± 2.5% inhibition, respectively (TNF- was not tested). These studies suggest that the upregulation of ERK1/2 and p38 isoforms was associated with cytokines that induced MIP-3/CCL20 release and was necessary for cytokine-induced secretion.

View larger version (21K): [in this window] [in a new window]   Figure 5. Inhibition of MAPK and MIP-3CCL20. To test the importance of ERK1/2 and p38 activation on MIP-3/CCL20 release, BEAS-2B cells were stimulated with IL-1, IL-4, or IL-13 after pretreatment with the MEK inhibitor PD 98059 or SB203580 to inhibit p38. Data are presented as mean ± SEM (n = 3; *P < 0.05 versus stimulated). Black bars, stimulus; lightly shaded bars, PD; darkly shaded bars, SB.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

In these studies, we described the ability of primary culture human AECs and BEAS-2B cells to synthesize and secrete MIP-3/CCL20. We demonstrated the release of this chemokine in response to low concentrations of a diverse group of cytokines, including the pro-inflammatory agents TNF- and IL-1ß and the Th2 cytokines IL-4 and IL-13. Furthermore, we demonstrated that stimuli, such as ambient particulate matter, induced the synthesis and release of this CC chemokine. The signaling pathways that induce the release of MIP-3/CCL20 remain incompletely described. Our data also demonstrated that this diverse group of stimuli was capable of upregulating ERK1/2 and p38 MAPK pathways and that the upregulation of these pathways was associated with MIP-3/CCL20 release.

MIP-3/CCL20 is a recently described CC-chemokine, first identified by a computer search using expressed sequence tags from complementary DNA libraries from public sequence databanks (9–11). MIP-3/CCL20 is also known as LARC (liver and activation-regulated chemokine) and Exodus-1. MIP-3/CCL20 shares structural similarity with some ß-defensins, which are also capable of functional binding to CCR6 (28). In contrast to the promiscuity between most chemokines and chemokine receptors, MIP-3/CCL20 is the unique chemokine ligand for the CC-chemokine receptor CCR6, a receptor that has a distinct pattern of expression. CCR6 has a limited distribution in DCs and has been described on some immature DCs but not in CD14⁺ DC precursors or mature DCs (29, 30). The restricted expression of CCR6 is consistent with the finding of MIP-3/CCL20 as the most potent chemokine to induce selective migration of in vitro-generated CD34⁺ hematopoietic progenitor cell-derived LC precursors and skin LC (29).

Because of the restricted expression of CCR6 and the selective interaction between MIP-3/CCL20 and CCR6, the pattern of expression of MIP-3/CCL20 is critical for DC recruitment (9–11). MIP-3/CCL20 has also recently been described in inflamed epithelial crypts of tonsils, epidermal keratinocytes in psoriatic and atopic dermatitis lesions, and inflamed human colonic epithelial cells (15, 17, 18, 29, 31, 32). In tonsillar crypts, its expression is restricted to discrete epithelial cells lining the lumen (18). Thus, its expression has been described at mucosal and epithelial surfaces. Our data extend these observations and describe its stimulated expression by primary culture and by transformed HBECs, suggesting a role for this chemokine in airway mucosal adaptive immune responses.

MIP-3/CCL20 is rarely constitutively expressed but requires stimulated expression. The mechanisms by which the gene and protein for MIP-3/CCL20 are upregulated are not completely described. The discrete stimuli that lead to its upregulation may be cell specific. In the monocytoid cell line J774, expression is induced by LPS but not by cytokines such as TNF-, IFN-, IL-1ß, or IL-4 (15). LPS also induces mLARC in intestinal tissues (15). In human intestinal epithelial cell lines, MIP-3/CCL20 is also upregulated in response to TNF-, IL-1ß, and enteric bacterial pathogens Salmonella or Escherica coli (17). Neutrophils and keratinocytes upregulate MIP-3/CCL20 in response to TNF- and IL-1ß and to LPS (31, 33, 34). Our data demonstrate a broad spectrum of cytokines that are capable of upregulating MIP-3/CCL20 inHBECs. These cytokines include those involved in inflammatory responses, and, in a manner similar to that of intestinal tissues, we noted MIP-3/CCL20 stimulation with TNF- and IL-1ß. Receptors for these cytokines have been well described in AEC, and we and others have demonstrated functional responses to these agents. IL-4 and IL-13 are Th2 cytokines with extensive overlap of biologic activities. IL-4R expression has been described in human AECs (35, 36). Elevated levels of IL-4 and IL-13 have been found in the lung with allergen provocation and in asthma (37). We have demonstrated the ability of these Th2 cytokines to upregulate MIP-3/CCL20 release by AEC at low concentrations. The data suggest that MIP-3/CCL20 may also be upregulated in Th2-associated diseases such as asthma.

Because of the recent interest in the health effects of PM, particularly in diseases such as asthma, we investigated whether ambient urban PM was capable of upregulating MIP-3/CCL20 (38). We used size-fractionated ambient PM collected from an urban environment as our model system. This model is a relevant environmental model because of the ambient source of PM. The particle sizes tested were all <3.2 µm, correlating most closely with the PM_2.5 fraction that is commonly monitored. In the urban environment, these particles are derived from mobile and regional sources and include diesel and other components (39). Our studies demonstrated the upregulation of MIP-3/CCL20 in AEC with particles of small sizes. The effect was specific for ambient particles because carbon particles failed to induce upregulation. This finding provides a further mechanism by which ambient particles, including diesel particles, may modify immune responses in the airway.

The intracellular signals associated with the upregulation of MIP-3/CCL20 are incompletely described. The response to pro-inflammatory cytokines such as TNF- makes MIP-3/CCL20 a likely target for NF-B regulation. This association has been demonstrated in intestinal epithelial cells with the suppression of MIP-3/CCL20 release in the presence of a recombinant adenovirus expressing a mutant IB protein (17). Moreover, overexpression of p65 activates the LARC (MIP-3/CCL20) promoter in intestinal epithelial cells and liver cells (40, 41). Our data demonstrated the upregulation of MIP-3/CCL20 by a broad spectrum of cytokines and by ambient particles. Because we and others have demonstrated the activation of MAPK pathways by TNF- and IL-1ß and by ambient PM (25, 26), we investigated whether the activation of MAPK pathways was associated with and necessary for MIP-3/CCL20 release. We focused on two MAPK pathways, ERK1/2 and p38. We first confirmed that each of the cytokines studied was capable of upregulating ERK1/2 and p38 in AEC and demonstrated rapid (15 min) and persistent (60 min) upregulation of ERK1/2 and p38.

Activation of the ERK and p38 MAPK pathways by TNF- and IL-1 has been well described. In most cells, ERK pathways are activated by Ras via Raf groups of MKKK, and p38 MAPK are activated by Rho family GTPases (27). Less is known about MAPK activation in response to IL-4 and IL-13. IL-4 signals via activation of IL-4R, which comprise dimers of IL-4-chain (IL-4) and -chain. IL-4R triggers the activation of receptor-associated Janus kinase proteins and the phosphorylation of STAT proteins, specifically STAT6 (42). A heterodimer of IL-13R1 and IL-4R (Type II IL-4R) also functions as a high-affinity receptor for IL-13 and IL-4. In airway smooth muscle cells, IL-4 and IL-13 cytokines have recently been demonstrated to activate ERK1/2 (43, 44). We extended these observations to AECs and demonstrated activation of ERK1/2 and p38 in response to IL-4 and IL-13 at a concentration that induced MIP-3/CCL20 release. These data show that discrete stimuli, each capable of activating different upstream signals, converge on ERK1/2 and p38. Moreover, this convergence has functional effects on MIP-3/CCL20 release.

In summary, these data describe the synthesis and release of the CC-chemokine, MIP-3/CCL20 by AEC. The wide range of stimuli, including pro-inflammatory cytokines, Th2 cytokines, and ambient PM, suggest that this chemokine may serve an important role in airway responses. The selective function of this chemokine, mediated by the specificity of ligand binding and the select expression of CCR6 on specific immature DCs, suggests a critical role in DC-associated airway diseases such as asthma.

	Acknowledgments

 
The authors thank Dr. Robert Norman for help in the preparation of this manuscript. These studies were supported by grants from the National Institutes of Health ES10187 (J.R.), T3207267, NCRR M01RR00096, ES00260 and from the Environmental Protection Agency R827351 and R826244 (T.G.).

Received in original form June 29, 2002

Received in final form October 11, 2002

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