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Originally published online as doi:10.1189/jlb.0305147 on April 14, 2005

Published online before print April 14, 2005
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(Journal of Leukocyte Biology. 2005;78:158-166.)
© 2005 by Society for Leukocyte Biology

Characterization of CCL20 secretion by human epithelial vaginal cells: involvement in Langerhans cell precursor attraction

Magali Cremel*, Willy Berlier*, Hind Hamzeh*, Fabrice Cognasse*, Philip Lawrence*, Christian Genin*, Jean-Claude Bernengo{dagger}, Claude Lambert{ddagger}, Marie-Caroline Dieu-Nosjean§ and Olivier Delézay*,1

* Groupe Immunité des Muqueuses et Agents Pathogènes (GIMAP), Faculté de Médecine J. Lisfranc, St Etienne, France;
{dagger} Centre Commun de Quantimétrie, Université Claude Bernard Lyon 1, France;
{ddagger} Laboratoire d’Immunologie Clinique, CHU, St Etienne, France; and
§ INSERM U255, Centre de Recherches Biomédicales des Cordeliers, Paris, France

1 Correspondence: GIMAP, Faculté de Médecine J. Lisfranc, 15 rue Ambroise Paré, 42023 St Etienne Cedex 2, St Etienne, France. E-mail: delezay{at}univ-st-etienne.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mucosa represents the main site of pathogen/cell interactions. The two main types of cells forming the epithelial structure [epithelial cells and Langerhans cells (LC)] coordinate the first defense responses to avoid infection. To evaluate the involvement of epithelial cells in the early steps leading to a specific adaptive immune response, we have studied the interactions between vaginal epithelial and LC through the establishment of a human vaginal epithelial mucosa. We demonstrate that normal human vaginal epithelial cells constitutively secrete the chemokine macrophage inflammatory protein 3{alpha}/CC chemokine ligand 20 (CCL20), known to recruit LC precursors (LCps) selectively via its cognate CC chemokine receptor 6 (CCR6). This secretion is up-regulated by the proinflammatory cytokine interleukin-1ß through the nuclear factor-{kappa}B pathway. Similar results were obtained with the human vaginal epithelial cell line SiHa, which displays numerous homologies with normal vaginal cells. The chemotactic activity of the secreted CCL20 was demonstrated by its ability to attract LCp CCR6+. Moreover, the use of neutralizing polyclonal antibodies directed against the CCL20 molecule abolished this migration completely, suggesting that CCL20 is the main attracting factor for LCps, which is produced by the vaginal cells. These data indicate that vaginal epithelial cells play an important role in the immunological defense by attracting immune cells to the site of epithelial/pathogen contact.

Key Words: chemokine • vaginal epithelial cells • mucosal model


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mucosal surfaces are exposed continuously to bacteria or viruses and represent a potential site of entry for numerous pathogens. Sentinel cells, including epithelial cells and Langerhans cells (LC), sense the environment and coordinate defenses for the protection of mucosal tissues [1 , 2 ]. This coordination is controlled, at least in part, by the secretion of soluble factors that allow innate immune responses to prevent and contain infection from highly diverse pathogens. During the course of inflammation or microbial infection, epithelial cells can respond to proinflammatory mediators such as tumor necrosis factor {alpha} (TNF-{alpha}) or interleukin (IL)-1ß, resulting in an influx of immune cells such as neutrophils, T cells, and LC precursors (LCps). The regulation of LCp recruitment by epithelial cells inside the mucosa is controlled by macrophage inflammatory protein 3{alpha}/CC chemokine ligand 20 (CCL20) secretion [3 , 4 ]. This chemokine, a protein of 70 amino acids, is the most potent chemoattractive molecule for LCps at the mucosal level and acts via its cognate CC chemokine receptor 6 (CCR6) [5 ]. CCL20, produced at sites of inflammation, may chemoattract CCR6-expressing LCps to the subepithelial region of mucosal surfaces [6 ]. As LC capture antigen at the mucosal surface, they undergo a functional and phenotypic change that includes a decrease in CCR6 expression and a concomitant increase in expression of CCR7, the receptor for secondary lymphoid tissue chemokines (CCL19 and CCL21) [7 ]. This change promotes LC emigration from the mucosa to the draining lymphoid organs (lymph nodes) toward the blood or lymphatic vessels. CCL20 is constitutively secreted in numerous normal human tissues including colon, skin, lung, and testis and is involved in the permanent recruitment of LCps in these epithelial sites [8 9 10 11 12 ].

The epithelial/LCps cell-cell interactions through chemokine secretion finely regulate innate immunity, confirming the important role of the CCL20 chemokine and the coordination of defenses of mucosal tissues. Deregulation of CCL20 secretion has been described in chronic inflammatory diseases at the mucosal sites, such as psoriasis [13 , 14 ], atopic dermatitis [15 ], and Crohn’s disease [10 , 16 ].

Although many studies have been performed on skin keratinocytes or on intestinal epithelial cells, enlightening the importance of CCL20 in LCp recruitment and its role in mucosal immunity, there is no study demonstrating the involvement of this chemokine in LCp recruitment in the human vaginal mucosa. The studies herein describe the constitutive and regulated CCL20 secretion by an in vitro vaginal epithelial mucosal model and demonstrate the capacity of this chemokine to attract LCps via its cognate receptor CCR6. This point could be of particular relevance for the study of pathogen transmission during sexual intercourse, and in particular, it could explain an increased risk of pathogen transmission in inflammatory conditions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Normal human vaginal cells (NHVC) were obtained from women undergoing routine hysterectomies as already described [17 ]. Briefly, biopsies (2–5 mm2) were washed several times with phosphate-buffered saline (PBS) containing an antibiotic-antimycotic solution (penicillin-streptomycin-amphotericin B, Sigma-Aldrich, St. Louis, MO) and were incubated with dispase II (Roche Diagnostics, Basel, Switzerland). Purified epithelial cells were washed with PBS and expanded in a 75-cm2 culture flask in keratinocyte serum-free medium (Invitrogen, Carlsbad, CA) supplemented with 5 ng/ml recombinant human (rh) epidermal growth factor (Invitrogen), 1 mM sodium pyruvate (Invitrogen), 2 mM L-glutamin (Invitrogen), and antibiotic-antimycotic solution (Sigma-Aldrich). Cells were used after 2 or 3 weeks when confluence was obtained.

The SiHa cell line was routinely grown in Dulbecco’s modified Eagle’s medium (DMEM)-F12 (Cambrex BioScience, Verviers, Belgium), supplemented with 10% fetal bovine serum (FBS) and antibiotic-antimycotic solution. To obtain a model of vaginal multilayer epithelium, the cells were seeded on precoated MatrigelTM filters (BD BioCoatTM MatrigelTM invasion chamber, BD Biosciences, Franklin Lakes, NJ) at a density of 400,000 cells/ml and cultured for 12 days.

LC were obtained from umbilical cord blood CD34+ hematopoietic progenitor cells. First, cord blood mononuclear cells were separated from granulocytes and erythrocytes by a Ficoll density gradient centrifugation. Monocyte depletion was performed by differential cell adherence in a 75-cm2 culture flask with RPMI-1640 (Cambrex BioScience) medium, completed with penicillin, streptomycin, amphotericin B, and 10% FBS. CD34+ progenitors were isolated throughout a positive immunoselection procedure (CD34 MultiSort kit, Miltenyi Biotec, Bergisch Gladbach, Germany). Cells were cultured in the presence of stem cell factor (10 U/ml, Peprotech, London, UK), granulocyte macrophage-colony stimulating factor (200 U/ml, Peprotech), TNF-{alpha} (50 U/ml, Peprotech), and 2.5% human AB+ serum in RPMI-1640 medium.

Cells were used at day 6 (LCps) for migration experiments and at day 12 (LC) for the study of the integration in the epithelial cell multilayers.

Flow cytometry
Epithelial cells were tested for intracellular cytokeratin (CK) expression after fixation with paraformaldehyde (3.6%, 15 min) and permeabilization (0.2% Triton X-100, 20 min). Cells were incubated with 2 µg mouse monoclonal antibody (mAb) anti-human CK4 (clone 6B10, Sigma-Aldrich), anti-human CK10 (clone K8.6, Sigma-Aldrich), anti-human CK13 (clone KS-1A3, Sigma-Aldrich), or anti-human CK14 (clone CKB1, Sigma-Aldrich). Cells were then incubated with 5 µg fluorescein isothiocyanate (FITC)-conjugated anti-mouse antibodies (DakoCytomation, Glostrup, Denmark). The efficiency of the permeabilization was evaluated by propidium iodide (PI) staining (1 µg/ml). A mouse anti-human leukocyte antigen (HLA)-I mAb (DakoCytomation) was used as positive control.

To quantify their integration rate in the epithelial multilayer, LC were labeled with 5.5 µg/ml carboxyfluorescein diacetate, succinimidyl ester (CFSE; Vybrant CFDA SE cell tracer, Molecular Probes, Eugene, OR) before their integration and were counted by flow cytometry after trypsinization of the multilayer.

Confocal microscopy
CFSE-labeled LC were added to the SiHa multilayer grown on permeable filters (BD BioCoatTM MatrigelTM invasion chamber, 8 µm pore size) for 1 h, 3 h, and 24 h. After washings, the multilayer was fixed for 30 min with 3.6% paraformaldehyde and permeabilized for 30 min with 0.2% Triton X-100. Nuclei were stained with PI (1 µg/ml in PBS 10% FBS). Filters were examined in PBS immersion using a confocal laser-scanning microscope system (Leica TCS SP2, Leica Microsystems, Wetzlar, Germany). Three-dimensional (3D) imaging was performed by reconstitution with the AmiraTM software (TGS Inc., San Diego, CA)

mRNA CCL20 detection
CCL20 mRNA was detected by reverse transcriptase-polymerase chain reaction (RT-PCR) after 4 h of cell treatment. The mRNA was extracted according to the manufacturer’s instructions with a GenElute direct mRNA miniprep kit (Sigma-Aldrich) and digested with RNase-free DNase (RQ1 DNase, Promega, Madison, WI) to eliminate DNA contaminations. mRNA (2 µL) was added to 18 µl of a mixture containing 200 U Moloney murine leukemia virus RT with 5x first-strand buffer (Invitrogen), 0.5 µg oligo-dT primer (Invitrogen), 0.5 mM deoxy-unspecified nucleoside 5'-triphosphate (dNTP; Roche Diagnostics), 10 mM dithiothreitol (Invitrogen), 40 U RNaseOUT (Invitrogen), and diethylpyrocarbonate (DEPC)-treated water. RT was performed for 1 h at 37°C.

A PCR targeting the open-reading frame of the CCL20 gene was performed with 3 µl cDNA, 1.2 U Taq polymerase with 10x buffer (Invitrogen), 3 mM MgCl2 (Invitrogen), 40 pmol CCL20 primers (forward: 5'-TTGCTCCTGGCTGCTTTG; reverse: 5'-ACCCTCCATGATGTGCAAG), 0.2 mM dNTP (Roche Diagnostics), and DEPC-treated water for a final volume of 50 µl. The thermocycling was performed on a Perkin Elmer 9600 thermocycler: 2 min at 95°C, 40 cycles of 1.5 min at 95°C, 1.5 min at 59°C, 1.5 min at 72°C, and a final elongation step of 7 min at 72°C. The CCL20 product [360 base pairs (bp)] was visualized under ultraviolet light after electrophoresis migration in a 1.5% (w/v) agarose gel and ethidium bromide staining. As control, the human gene of ß-actin was amplified by RT-PCR for each sample.

CCL20 detection and quantification
The production of CCL20 was quantified in the supernatants of SiHa cells and NHVC after 18 h of culture using an enzyme-linked immunosorbent assay (ELISA) detection kit (Quantikine, R&D Systems, Abingdon, UK), according to the manufacturer’s instruction. Optical density was measured at 450 nm. All assays were performed in triplicate.

Chemotaxis assay
Migration of LCps (tested for CCR6+ expression) was assayed by measuring their capacity to migrate across MatrigelTM-coated transwell inserts (BD BioCoatTM MatrigelTM invasion chamber, 8 µm pore size). Briefly, 500,000 LCps were added to the apical compartment of the filter (upper compartment) and the basal compartment (lower compartment) containing the medium to be tested. The number of migrated LCps found in the basal medium was determined by microscope observation and counting. CCL20 chemotactism inhibition was performed by using neutralizing polyclonal antibodies (10 µg/ml, R&D Systems), which were added to the cell supernatant 2 h before the chemotaxis assays or irrelevant antibodies as control.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Flow cytometry analysis of NHVC and SiHa cells
We first characterized the expression of different markers to evaluate the homology between NHVC and the SiHa cell line, as well as their state of cellular differentiation. As shown in Figure 1A , the expression of all markers tested was comparable for NHVC and the SiHa cells. The two cell types expressed the HLA-I molecule and the CK4, -10, -13, and -14. CK4, characteristic of differentiated epithelial cells (the superficial layers), was found to be expressed for 76.5% and 43.1% of NHVC and SiHa cells, respectively. Suprabasal cells, characterized by CK13 expression, were found to represent 67.3% and 78.8% of NHVC and SiHa cells, respectively. No difference was observed for the detection of the two other CKs (CK10 for differentiated cells and CK14 for proliferating cells, basal layers), which were expressed by all the cells. These data indicated that NHVC and the SiHa cell line grow as a multilayer, displaying the different states of cell differentiation.



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  		Figure 1. Comparison of CK expression between NHVC and the SiHa cell line by flow cytometry analysis. (A) Cells (SiHa, solid bars; NHVC, hatched bars) were fixed and permeabilized before incubation for 1 h with 2 µg different mAb directed against HLA, galacotosylceramide (GalCer), and CK4, -10, -13, and -14. After washing, 5 µg goat FITC-conjugated anti-mouse antibodies were used to visualize the labeling by flow cytometry on a Beckman-Coulter Epics XL cytometer. Irrelevant antibody (mouse immunoglobulin G, DakoCytomation) served as negative control. (B) The evolution of the median fluorescent intensity (mfi) associated with SiHa cells was evaluated during the cell culture time. Cells were cultured for 21 days on permeable support (precoated MatrigelTM filters) and regularly tested for CK expression by flow cytometry analysis as described above. All the results are representative of at least three independent experiments.

 
To study the kinetics of the differentiation of SiHa cells during cell culture, we analyzed the evolution of the labeling intensity (mfi) for the CKs described above (Fig. 1B) . No significant evolution of CK4, CK13, and CK14 was observed during cell culture. In contrast, the CK10 labeling pattern displayed a significant increase after 12 days of cell culture, suggesting the appearance of well-differentiated cells at this time.

These data suggest that our epithelial cell culture conditions allowed the establishment of a multilayer displaying the different states of cell differentiation found in vivo: a remaining proliferative cellular population (characterized by CK14 expression) with an increase in differentiated cell population (characterized by CK10 expression).

Integration and stability of LC in the SiHa multilayer
To study the interactions between LC and vaginal epithelial cells, CFSE-labeled LC were added to a multilayer of SiHa cells grown on a MatrigelTM-precoated filter. We first studied the kinetics of LC integration by confocal microscopy analysis. As shown in Figure 2A , LC (green) were found to be integrated inside the multilayer formed by SiHa cells (PI-labeled, red) after 3 h and were able to cross this epithelial structure after 24 h. The quantification of LC by flow cytometry in the three compartments defined by the filter (apical, cellular multilayer, or basal compartment) confirmed our first observations by confocal analysis, demonstrating the rapid integration of LC and their ability to cross the multilayer after 24 h (Fig. 2B) . It should be noted that the amount of LC integrated inside the multilayer remained stable (~12,000 cells, representing 2% of the total cells found in the multilayer), in agreement with the in vivo situation. In another experiment, the number of LC remaining inside the SiHa multilayer was determined from 24 to 96 h after their addition to the SiHa multilayer. As shown in Figure 2C , the number of LC incorporated inside the SiHa multilayer was stable during the time of cell culture even after 96 h (between 17,000 and 21,000 cells). Moreover, microscopy observations of the LC after trypsin disruption of the multilayer revealed that these cells remained viable after 96 h inside the multilayer. This result suggested specific LC/SiHa cells interactions that permit the homing and the viability of the immune cells inside the multilayer.



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  		Figure 2. Kinetics of LC integration inside the SiHa epithelial multilayer. (A) CFSE-labeled (green) LC (derived from CD34+ hematopoietic cell progenitors) were added to the SiHa epithelial cell multilayer cultured on precoated MatrigelTM filters (permeable support) for 1, 3, or 24 h. After washing steps, the cells were fixed and incubated with PI (1 µg/ml) to visualize the cell nucleus (red). Confocal analysis was performed by using a Leica microscope, and the 3D reconstitution was realized with the AmiraTM software. Original bar, 20 µm. (B) Apical mediums (upper compartment) and basal mediums (lower compartment) of the filters described in A were collected. The multilayers growing on the filters were disrupted by tryspin treatment. The three compartments, apical (hatched bars), multilayer (solid bars), and basal (open bars), were analyzed for CFSE-labeled LC content by flow cytometry. (C) The number of CFSE-labeled LC integrated inside the SiHa multilayer (solid bars) was quantified during the period of cell culture by flow cytometry 24, 48, 72, or 96 h after LC deposition on the apical compartment of the filter. The number of LC found in the basal compartment was also evaluated (grey bars). (A–C) All results were representative of three independent experiments.

 
CCL20 production by human epithelial vaginal cells
As it has already been demonstrated that epithelial cells could attract LCps via CCL20 production [4 , 8 ], we analyzed the secretion of this chemokine by NHVC and by the SiHa cell line under different culture conditions.

In this experiment, the supernatants of NHVC and SiHa cells were tested for CCL20 content by using a quantitative ELISA assay (Fig. 3A ). NHVC and SiHa were able to accumulate significant amounts of CCL20 in their supernatants without any stimulation (184±2 pg/ml and 167±24 pg/ml, respectively, after 17 h). After IL-1ß treatment (25 ng/ml), the level of CCL20 secretion found in the two supernatants was increased dramatically (697±5 pg/ml and 822±25 pg/ml for NHVC and SiHa cells, respectively). This data confirmed the constitutive production of CCL20 by human epithelial vaginal cells and demonstrated that these cells were sensitive to proinflammatory stimulation, resulting in the increase of CCL20 secretion and accumulation in the cell supernatant. The pretreatment of SiHa cells with BAY 11-7085, an inhibitor of the NF-{kappa}B intracellular pathway, resulted in a complete inhibition of IL-1ß-induced CCL20 production. As shown in Figure 3B , the level of CCL20 accumulation in the supernatant of SiHa cells reached 39 ± 8 pg/ml (for untreated cells), 339 ± 48 pg/ml (for IL-1ß-treated cells), and 20 ± 3 pg/ml (for IL-1ß treatment after BAY 11-7085 preincubation). The effect of BAY 11-7085 was not associated with cell death, as preliminary cytotoxicity assays were carried out without significant cell mortality at this concentration (data not shown).



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  		Figure 3. CCL20 secretion by the vaginal epithelial cells. (A) Supernatants of NHVC or SiHa (untreated, open bars; stimulated with 25 ng/ml IL-1ß, solid bars) cultured on plastic support were collected after 17 h and analyzed for CCL20 content by a quantitative immunoassay (Quantikine ELISA, R&D Systems). The results are expressed as the means of triplicates ± SD. (B) SiHa cells cultured on plastic support and stimulated overnight with 25 ng/ml IL-1ß in the absence or presence of 2.5 µg/ml of a specific nuclear factor (NF)-{kappa}B pathway inhibitor (BAY 11-7085, Tebu). Supernatants of untreated cells were tested as controls. The results are expressed as the means of three different experiments ± SD. (C) SiHa cells, cultured on permeable support (BD BioCoatTM MatrigelTM invasion chamber, 8 µm pore size) were stimulated for 16 h with 25 ng/ml IL-1ß. Apical (upper compartment) and basal (lower compartment) mediums were tested for CCL20 content by ELISA (Quantikine ELISA, R&D Systems). The results are expressed as the means of triplicates ± SD.

 
When the cells were cultured on permeable support (precoated MatrigelTM filters), the CCL20 production was mainly found in the apical compartment, suggesting a polarization of the chemokine secretion and the establishment of a CCL20 concentration gradient leading to the attraction of CCR6+ cells toward the vaginal surface (Fig. 3C) .

CCL20 mRNA detection in SiHa cells
To study the regulation of CCL20 secretion, RT-PCR analysis was performed on SiHa cells after different stimulations (Fig. 4 ). The results indicated that the increase in CCL20 secretion induced by IL-1ß treatment was a result of an increase in CCL20 mRNA production by the cells. In the absence of treatment (Fig. 4 , lane 1), a weak specific signal of CCL20 mRNA (360 bp product) was detected, suggesting a low constitutive expression of mRNA, in agreement with CCL20 secretion. However, the overnight treatment of the SiHa cells with IL-1ß (25 ng/ml, Fig. 4 , lane 2) or TNF-{alpha} (50 ng/ml, Fig. 4 , lane 5) resulted in an increase in the specific signal. In contrast, no signal variation could be detected with TGF-ß (25 ng/ml, Fig. 4 , lane 4). The strongest signal was obtained with IL-1ß. This signal was attenuated when the cells were pretreated with a specific inhibitor of the NF-{kappa}B intracellular cascade (BAY 11-7085, 2.5 µg/ml, Fig. 4 , lane 3) before IL-1ß stimulation, confirming the results obtained above for CCL20 secretion.



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  		Figure 4. CCL20 mRNA expression by SiHa cells. Confluent SiHa cells were untreated (lane 1) or treated for 4 h with IL-1ß (25 ng/ml, lane 2), IL-1ß (25 ng/ml) after BAY 11-7085 pretreatment (2.5 µg/ml, 1 h, lane 3), transforming growth factor-ß (TGF-ß; 25 ng/ml, lane 4), and TNF-{alpha} (50 ng/ml, lane 5), before the mRNA extraction process. CCL20 RT-PCR was performed on all the extracts. The specific band (360 bp) was visualized by ethidium bromide staining on a 1.5% agarose gel. Lane 6, Internal PCR negative control. M, Molecular weight markers. Results were representative of three independent experiments.

 
Migration of CCR6+ LCps
Another series of experiments were conducted to analyze the functionality of the CCL20 secreted by the SiHa vaginal epithelial cell line. The chemoattractive effect of CCL20 on LCps expressing CCR6 (CCR6+ LCps) was evaluated by analyzing the effect of rhCCL20 or supernatants of SiHa cells on CCR6+ LCp migration across an 8-µm pore MatrigelTM-precoated filter (BD BioCoatTM MatrigelTM invasion chamber). For these experiments, CCR6-positive LCps (more than 80% of the cells expressed CCR6, data not shown) were applied to the apical compartment of an invasion chamber in RPMI medium The basal compartment was filled with different mediums (DMEM-F12 as control, rhCCL20, or SiHa cell supernatants). After 20 h, the number of cells found in the basal compartment was determined. As demonstrated in Figure 5A , rhCCL20 (50 ng/ml) was able to stimulate CCR6+ LCp migration, as the number of LCps found in the basal compartment was enhanced compared with DMEM-F12 medium alone (6083±1217 cells vs. 38,910±114 cells, respectively, for DMEM-F12 and rhCCL20). Similarly, supernatants of untreated SiHa cells were able to induce CCR6+ LCp migration (136,035±8100 cells found in the basal compartment). Furthermore, this migration was enhanced significantly with the supernatant of IL-1ß-treated cells (195,342±4624 cells). As IL-1ß was not directly involved in cell migration (data not shown), these data confirmed that CCR6+ LCps were able to migrate to a CCL20-containing compartment and that the CCL20 secreted by SiHa cells (under basal conditions or after IL-1ß stimulation) was active. It should be noted that the chemoattraction properties of the SiHa supernatants were more efficient than a concentration of 50 ng/ml CCL20, suggesting an increased efficiency of the CCL20 secreted by epithelial cells than the rhCCL20. However, we could not exclude the presence of other chemoattractive molecules also secreted by the SiHa cells in these supernatants. To elucidate this point, another experiment was conducted by using polyclonal neutralizing antibodies directed against CCL20 or irrelevant polyclonal antibodies as control (10 µg/ml). As shown in Figure 5B , the preincubation of SiHa cell supernatants (from untreated or IL-1ß-treated cells) with these anti-CCL20-neutralizing polyclonal antibodies resulted in a dramatic decrease of CCR6+ LCp migration performed during 6 h. In the presence of these antibodies, the number of LCps that migrated to the basal compartment was reduced to 1250 ± 250 cells compared with 4500 ± 500 cells without antibodies for the supernatant of untreated cells and reduced to 2250 ± 750 cells compared with 10,250 ± 250 cells for the supernatant of IL-1ß-treated cells. The use of an irrelevant antibody in the same conditions was ineffective for the neutralization of LCp migration (with or without IL-1ß treatment), confirming the specificity of CCL20 attraction (Fig. 5B) . These results confirmed that CCL20 was the main chemoattractive molecule present in the vaginal epithelial cell supernatants able to induce LCp migration.



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  		Figure 5. CCL20 attraction of CCR6+ LCps. (A) CCR6+ LCps were added to the apical compartment of MatrigelTM-coated invasion filters (BD BioCoatTM MatrigelTM invasion chamber, 8 µm pore size) and were tested for their ability to migrate over 20 h to the basal compartment containing different SiHa cell supernatants (treated with 25 ng/ml IL-1ß or untreated). Medium alone or rhCCL20 (50 ng/ml) in the basal compartment served as negative and positive controls for CCR6+ LCp migration, respectively. Values reported are expressed as the mean ± SD of three experiments. (B) Inhibition of CCL20-mediated LCp migration. As described above, supernatants from untreated or IL-1ß-treated (25 ng/ml) SiHa cells were tested for their ability to attract CCR6+ LCps over 6 h with or without preincubation with polyclonal-neutralizing CCL20 antibodies (ab) or irrelevant polyclonal antibodies (10 µg/ml, R&D Systems). The results are expressed as the means ± SD of two experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we have analyzed the interactions between epithelial and LCps of the vaginal mucosa to evaluate the role of epithelial cells in the early stages of the immunological responses through CCL20 secretion. To this end, we have used the SiHa vaginal epithelial cell line displaying numerous morphological and biochemical similarities with epithelial NHVC [18 ]. Confirming these data, the comparison between SiHa cells and epithelial NHVC, obtained from hysterectomies, does not show significant differences for all the markers tested. The SiHa cells are able to grow as a multilayer and express the CKs characteristic of the different degrees of cell differentiation [19 ]. Indeed, the SiHa multilayer grown on MatrigelTM support is representative of the in vivo vaginal situation, with proliferative epithelial cells and more superficial, differentiated ones [17 ]. The addition of LC (12-day-cultured cells obtained from hematopoietic progenitor CD34+) to the SiHa multilayer demonstrated the ability of these immune cells to integrate and remain viable inside the multilayer for 96 h in the same proportion as that observed in vivo [20 ]. This experiment also demonstrated that these LC (expressing the Langerin marker but not the CCR6) could cross our multilayer model.

To evaluate the role of epithelial cells in the recruitment of LCps and their steady-state in the vaginal pluristratified structure, we analyzed the production of CCL20 (the most potent chemoattractive molecule for LCps) by our vaginal epithelial cells. Our results demonstrate that SiHa cells were able to secrete the chemokine in the same order as the NHVC. This secretion was increased significantly by the proinflammatory cytokine IL-1ß through the NF-{kappa}B pathway, as demonstrated by the use of the specific inhibitor BAY 11-7085, and was mainly polarized at the apical side (mimicking the vaginal lumen) of the multilayer. These results were confirmed by the analysis of CCL20 mRNA synthesis, as we evidenced a strong increase in mRNA expression after the treatment of the SiHa cells with IL-1ß (or TNF-{alpha}). The mRNA signal returned to the basal level when the cells were treated with BAY 11-7085 before IL-1ß stimulation. This result, in perfect agreement with those obtained for CCL20 secretion, confirmed the involvement of the NF-{kappa}B intracellular pathway [21 , 22 ]. These data are closely related to those obtained with other epithelial cells [3 , 6 , 23 ]. In addition, the functional activity of the CCL20 secreted by SiHa cells was demonstrated by its capacity to specifically attract CCR6+ LCps.

In the absence of inflammation or antigenic stimulation, the low constitutive production of CCL20 may serve to maintain LCps (and/or other CCR6+ cells, such as memory T cells) inside the multilayer or in the close proximity of the vaginal lumen. In response to inflammatory stimuli, vaginal epithelial cells may develop the capacity to chemoattract LCps, via CCL20, toward the top of the epithelial structure, the vaginal lumen.

Taken together, our results demonstrate that vaginal epithelial cells participate actively in the immune defense by recruiting CCR6+ cells, and among them, the antigen-presenting cells (APC), which are important in the host adaptative immune response. This point is of particular importance if we consider that the vaginal mucosa is continuously exposed to bacteria or viruses that could activate epithelial cells and induce CCL20 secretion. In this way, it has been demonstrated that bacterial compounds [24 ] or viruses [25 ] could induce CCL20 secretion by numerous epithelial cells and then attract LCps. This idea suggests a fine regulation of interactions between epithelial and immune cells by chemical factors (such as cytokines and chemokines) that directly influence the responsiveness of the mucosal immune system, determining an adaptive immune response [26 ]. The role of the epithelial cells as gatekeepers of innate immune protection through the secretion of cytokines (IL-6, IL-8) has recently been described for cells isolated from the endometrium or Fallopian tube [27 ], confirming the involvement of epithelial cells in the homeostasis of immune cells throughout the human female tract.

The deregulation of such interactions, particularly concerning CCL20 secretion, could result in chronic inflammation diseases such as intestinal bowel disease [16 ], rheumatoid arthritis [28 ], psoriasis [13 ], atopic dermatitis [15 ], as well as several epithelial cancers (breast, cervix, pancreas, liver, and thyroid [29 , 30 ]). Moreover, the stimulatory effect of IL-1ß (as well TNF-{alpha}: cytokines that can be produced by mononuclear cells during acute inflammation) on vaginal epithelial CCL20 secretion could also explain, at least in part, the increased efficacy of pathogen transmission during sexual contact. Indeed, the inflammatory status of the vaginal mucosa could be responsible for a more efficient transmission of sexually transmitted diseases, in particular, for human immunodeficiency virus (HIV) transmission [31 , 32 ], probably by a more efficient attraction of immune cells to the vaginal mucosa. As APC, which are naturally present in the vaginal mucosa, could interact with HIV particles [33 ] and transport them to lymph nodes [2 ], the attraction of such cells to the site of HIV/epithelial cell contact during sexual intercourse could be responsible for the first events involved in sexual HIV transmission, the most common mode of contamination for this virus.

As it is well-assumed that CCL20 can play a crucial role in T cell or LCp recruitment, the secretion of other chemoattractive molecules by epithelial cells under inflammatory stimulations could not be excluded. In this way, it has been demonstrated that the antimicrobial peptide, human ß-defensin 2 (hBD-2), could be secreted by epithelial cells in response to proinflammatory cytokine stimulations or bacterial infections [34 ]. This molecule could also interact, but to a lesser extent, with CCR6, attracting immune cells expressing this receptor [35 ]. However, in our experiments, it seems that CCL20 is the main chemoattractive factor found in vaginal epithelial cell supernatant, as anti-CCL20 polyclonal antibodies completely inhibit LCp chemoattraction. hBD-2 could participate in the immune vaginal response by its antimicrobial rather than chemoattraction properties. This idea suggests that the stimulation of vaginal epithelial cells by proinflammatory cytokines or pathogens induces two main types of epithelial cell response: a direct effect on the pathogen by defensins and a selective LCp recruitment by CCL20.

The microenvironment of the vaginal epithelium is far more complex and includes many other diverse cells than epithelial cells, such as macrophages or lymphocytes, in addition to LCps. However, our epithelial vaginal model, constituted of SiHa human epithelial cells and LCps, seems to be a valuable tool for the evaluation of numerous compounds that could influence the vaginal mucosal response. In this way, the screening of mucosal adjuvants on SiHa cells could be based, at least in part, on the ability of the cells to secrete CCL20 (further experiments to evaluate this point are currently ongoing in our lab). Moreover, the possibility to substitute LC for other immune cells usually found in the vaginal mucosa in vivo (macrophages, lymphocytes) could permit the study of epithelial cell/immune cell interactions through direct cell/cell contact (coculture) or soluble factors (cell supernatants), as demonstrated in this study for LCps/vaginal cell interactions. Indeed, the adjunction of different cellular populations may allow numerous studies for a better understanding of the events that coordinate the cellular response to pathogenic stimulations. Finally, this vaginal epithelial model could be used to study the transepithelial crossing of numerous pathogens and in particular, for the study of heterosexual HIV transmission.


   ACKNOWLEDGEMENTS
 
This study was supported by the Agence Nationale de Recherches sur le SIDA (ANRS; Paris, France). W. B. is the recipient of an ANRS fellowship. We thank Chrystelle Meunier for helpful technical assistance and Dr. Fargier (Clinique Michelet, Saint Etienne, France) and Pr. Seffert (Gynecology Department, University Hospital, St Etienne, France) for cord blood and vaginal specimens.

Received March 15, 2005; accepted March 20, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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