Laboratory of Mucosal Immunology, Departments of Medicine and Pediatrics, University of California, San Diego, La Jolla, California
Submitted 1 April 2004 ; accepted in final form 22 September 2004
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ABSTRACT |
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CCL20; macrophage inflammatory protein-3; forskolin; G protein-coupled receptors; tyrosine phosphorylation
We and others recently reported that human intestinal epithelial cells express receptors for several members of the chemokine family (e.g., CXCR4, CCR5, CCR6, and CX3CR1) (4, 10, 24). Chemokine receptors are seven-transmembrane G protein-coupled receptors, and signaling through those receptors on leukocytes generally, but not exclusively, occurs through pertussis-sensitive Gi subunits (38). This also appears to be the case for CXCR4 and CX3CR1 expressed by intestinal epithelial cells (4, 10, 11, 24).
CCR6 is the sole known receptor for the chemokine CCL20 [also termed macrophage inflammatory protein-3 (MIP-3
) (41), liver and activation-regulated chemokine (LARC) (18), or Exodus-1 (21)]. CCR6 is expressed by immature myeloid lineage dendritic cells, which can migrate to the subepithelial region of mucosal surfaces in response to CCL20 stimulation, and by circulating memory T cells in humans that express the
4
7-integrin characteristic of mucosal homing lymphocytes (5, 9, 22, 30, 42, 51).
We recently noted that CCR6 is expressed by human colon epithelial cell lines and by human colonic epithelium in vivo (23). In response to bacterial infection or stimulation with proinflammatory mediators (e.g., TNF-, IL-1), intestinal epithelial cells upregulate mRNA expression and production of CCL20 protein as well as human
-defensin 2 (hBD2), an antimicrobial peptide that also has been reported to signal dendritic cells through CCR6 (51). Notably, CCL20 was secreted predominantly from the basolateral and not the apical membrane of polarized monolayers of intestinal epithelial cells (23). These studies raised the question as to whether CCL20 potentially mediates its effects on the intestinal epithelium as a paracrine/autocrine factor acting through basolaterally expressed CCR6 or whether the expression of CCR6 by intestinal epithelial cells is apically polarized and not likely to have contact with CCL20 in the presence of a normal epithelial barrier.
In the present study, we investigated whether CCR6 acts as a functional signaling receptor in intestinal epithelial cells and examined its distribution on those cells. We report that CCR6 expressed by intestinal epithelial cells is apically polarized and acts as a functional G protein-coupled receptor in these cells. CCL20 stimulation of CCR6-expressing human intestinal epithelial cells results in tyrosine phosphorylation of an adaptor/scaffolding protein, p130Cas, a key component of the pathway by which focal adhesion kinase (FAK) promotes cell migration. In addition, CCR6 stimulation results in the modulation of cAMP-stimulated electrogenic chloride secretion.
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MATERIALS AND METHODS |
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Human colon epithelial cell lines. The human colon adenocarcinoma cell lines HCA-7, Caco-2, and HT-29 were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 or 15% (Caco-2) heat-inactivated fetal bovine serum and 2 mM L-glutamine as described previously (10). T84 human colon carcinoma cells were grown in 50% DMEM-50% Ham's F-12 medium supplemented with 5% newborn calf serum and 2 mM L-glutamine (25). The cells were maintained in 95% air-5% CO2 at 37°C.
Flow cytometry.
Confluent T84, Caco-2, HCA-7, and HT-29 cells (1 x 106 cells) were detached with 20 mM EDTA in phosphate-buffered saline (PBS), fixed for 10 min at 4°C in 2% paraformaldehyde in PBS, and subsequently incubated for 90 min at 4°C with 10 µg/ml mouse anti-human CCR6 MAb or a mouse isotype-matched control antibody, followed by incubation for 1 h with R-phycoerythrin-labeled goat anti-mouse IgG. In other experiments, paraformaldehyde-fixed cells were permeabilized by inclusion of 0.1% saponin in the dilution and wash buffers. Cells were analyzed using flow cytometry (FACScan; Becton Dickinson, Sunnyvale, CA).
Confocal microscopy.
Polarized HCA-7 cells were cultured on microporous collagen-coated filter inserts (0.4-µm pore size, Transwell; Costar, Cambridge, MA) and allowed to grow for approximately 7 days as described previously (31), at which time they manifested a resistance of 500700 ·cm2. Cells were fixed in 4% paraformaldehyde in PBS for 10 min at 4°C, followed by incubation in PBS/1% bovine serum albumin (BSA) with 10% goat serum for 1 h at room temperature. Cells were then incubated at 37°C for 4 h with mouse anti-human CCR6 MAb (10 µg/ml) or a mouse monoclonal IgG2b isotype control applied from both the apical and basolateral sides. Cells were subsequently washed and incubated with Cy3-labeled goat anti-mouse IgG as secondary antibody and Alexa 488-coupled phalloidin at 4°C overnight. Filters were removed, mounted on glass slides, and examined using laser scanning confocal microscopy (MRC Bio-Rad 500; Bio-Rad Laboratories, Hercules, CA).
Immunoprecipitation and immunoblotting. HCA-7, Caco-2, and T84 cells were washed in DMEM or 50% DMEM-50% Ham's F-12 (T84) medium supplemented with 1% BSA and 2 mM L-glutamine, allowed to equilibrate for 1 h at 37°C, and then stimulated with CCL20 or PMA. Cells were washed in ice-cold PBS and lysed in ice-cold lysis buffer [10 mM Tris·HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.1% sodium deoxycholate, 1 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, and 0.5% protease inhibitor cocktail set III] for 30 min, sonicated, and spun at 14,000 rpm for 10 min, after which supernatants were assayed for protein content (Bio-Rad protein assay kit). Protein (20 µg/lane) was electrophoresed on 10% SDS-PAGE gels and transferred to nitrocellulose membranes (Hybond ECL; Amersham Pharmacia). Membranes were blocked with Tris-buffered saline (TBS) containing 10% nonfat dry milk, 1% donkey serum, and 0.1% Tween 20 and then incubated overnight at 4°C in a 1:1,000 dilution of rabbit antibodies against the different signaling molecules in dilution buffer (1% nonfat dry milk, 1% donkey serum, 0.1% Tween 20 in TBS). Blots were washed and incubated for 1 h with donkey anti-rabbit Ig-HRP. Alternatively, membranes were developed using biotin-conjugated sheep anti-rabbit IgG followed by streptavidin-HRP. Western blots were developed using enhanced chemiluminescence agents according to the manufacturer's instructions (Amersham) and were exposed to imaging film (XAR; Kodak).
For immunoprecipitation, equal concentrations of cell lysates were incubated overnight with anti-phosphotyrosine MAb (Tyr-100; 5 µg/ml), after which protein G-agarose was added. After centrifugation, pellets were extensively washed in lysis buffer, resuspended in 2x gel loading buffer [50 mM Tris (pH 6.8), 2% SDS, 200 mM dithiothreitol, 20% glycerol, and 0.2% bromphenol blue], and boiled before separation by SDS-polyacrylamide gel electrophoresis. Resolved proteins were transferred to a nitrocellulose membrane and probed with anti-p130Cas MAb followed by biotin-conjugated sheep anti-mouse IgG and streptavidin-HRP and then developed as described above. Total cell lysates of polarized HCA-7 cells stimulated either apically or basolaterally with CCL20 or PMA were incubated with anti-phosphotyrosine MAb, precipitated, transferred to nitrocellulose membranes, and probed with mouse anti-p130Cas followed by HRP-conjugated sheep anti-mouse IgG as described above.
cAMP assays. Confluent T84 cells in 12-well plates were washed with HEPES-buffered Ringer solution and incubated with 1 mM IBMX. After 10 min, CCL20 or somatostatin was added. After an additional 10 min, 1 µM forskolin, 0.1 µM PGE2, or 1 nM VIP was added, and cells were incubated for 10 min. In some cases, cells were pretreated with pertussis toxin (100 ng/ml) for 18 h before CCL20 stimulation, as indicated. cAMP levels in cell extracts were assayed using a cAMP enzyme immunoassay system (Amersham Pharmacia Biotech). In other experiments, confluent T84 cells were stimulated with CCL20 (100 ng/ml) for 90, 60, 30, 15, or 10 min before the addition of forskolin. IBMX (1 mM) was added 10 min before forskolin stimulation. Cells were incubated for 10 min after the addition of forskolin (1 µM), and cAMP levels were measured as described above. In time course studies, confluent T84 cells were treated for 10 min with CCL20, and cAMP levels were measured at 0, 5, 10, and 30 min after forskolin stimulation. To parallel conditions in the electrophysiological studies described below, the latter experiments were done in the absence of IBMX.
Electrophysiological studies. Vectoral ion transport was examined in modified Ussing chambers as described previously (26). The mucosal and serosal baths contained Ringer buffered salt solution supplemented with glucose (115 mM NaCl, 25 mM NaHCO3, 2.4 mM K2HPO4, 0.4 mM KH2PO4, 1.2 mM MgCl2, 1.2 mM CaCl2, and 10 mM glucose; adjusted to pH 7.4), which was gassed with 95% O2-5% CO2 at 37°C. The transepithelial voltage clamp connected to the Ussing chambers provided continuous monitoring of short-circuit current (Isc). Open-circuit potential difference was measured every 15 min, and transepithelial resistance was calculated using Ohm's law. After initial stabilization, baseline measurements of Isc, potential difference, and conductance were recorded for 40 min. Subsequently, agonists were added to the apical and basolateral baths to stimulate chloride secretion, and changes in electrophysiological parameters were recorded at regular intervals. These studies were done in the absence of IBMX.
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RESULTS |
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CCL20 inhibits forskolin-stimulated chloride secretion by intestinal epithelial cells. Increased electrogenic chloride secretion is an important functional consequence of increased cAMP levels in intestinal epithelial cells and is primarily responsible for promoting intestinal fluid secretion and secretory diarrhea. Therefore, we investigated whether CCL20 inhibition of forskolin-stimulated increases in cAMP were accompanied by decreased forskolin-stimulated epithelial cell chloride secretion, as assessed by alterations in Isc in Ussing chambers. Pretreatment of polarized T84 cells with CCL20 significantly reduced the peak Isc response to forskolin from 39 ± 5 µA/cm2 to 25 ± 6 µA/cm2, a decrease of 39 ± 7% (P < 0.05, n = 3 experiments) (Fig. 3B). The magnitude of this decrease was comparable to that seen after pretreatment of T84 monolayers with somatostatin (1 µM) before forskolin stimulation of electrogenic chloride secretion (39 ± 5%, P < 0.05, n = 3 experiments). These data indicate that CCL20 can function as a negative regulator of intestinal epithelial cell chloride secretion and are consistent with the observed decrease in cAMP production by CCL20-treated cells over the same time frame (Fig. 3A).
Because chemokines activate calcium fluxes in leukocytes (38) and calcium fluxes in intestinal epithelial cells also stimulate epithelial cell chloride secretion (45), in further studies we examined the ability of CCL20 to stimulate calcium fluxes in colonic epithelial cells. CCL20 stimulation did not affect baseline intracellular Ca2+ levels or basal Isc in T84 cells, which is consistent with the absence of calcium fluxes noted after stimulation with other chemokines (11) Although stimulation with a calcium ionophore, ionomycin, induced significant calcium fluxes in T84, Caco-2, and HCA-7 cells, stimulation with CCL20, as in T84 cells, induced little or no change in intracellular Ca2+ levels in Caco-2 or HCA-7 cells (data not shown), indicating that the inhibitory effects of CCL20 effects on epithelial chloride secretion are likely to be specific for the cAMP-mediated chloride secretory pathway.
CCL20 stimulates tyrosine phosphorylation of p130Cas.
To further investigate pathways by which CCR6 transduces signals into human colon epithelial cells, we stimulated HCA-7, T84, and Caco-2 cells with CCL20 and assessed the phosphorylation of Akt as an important downstream effector of signaling through phosphoinositide 3-kinase and the conventional PKC/
and novel PKC
isoforms as important signal transduction molecules downstream of the lipid second messenger diacylglycerol. CCL20 stimulation did not result in phosphorylation of Akt or the PKC isoforms, although Akt was phosphorylated by signaling through the EGF receptor and the PKC isoforms were activated by stimulation with phorbol esters (data not shown). We next assessed whether CCL20 increases tyrosine phosphorylation of cellular proteins. Interestingly, these studies revealed a marked increase in tyrosine phosphorylation of a protein with a molecular mass of
130 kDa within 1 min of CCL20 stimulation, which peaked by 510 min and began to decrease by 15 min (Fig. 4A).
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CCR6 expression is predominately apical in polarized colon epithelial cells. Intestinal epithelial cells are morphologically and functionally polarized in vivo. To assess the distribution of CCR6 on polarized intestinal epithelial cells, HCA-7 cells, grown as polarized monolayers on microporous filter supports, were immunostained for CCR6 and examined using confocal microscopy. As shown in Fig. 5, the most intense staining for CCR6 was in the apical region. A similar apical distribution of CCR6 was found in polarized T84 and Caco-2 cells (not shown).
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DISCUSSION |
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Electrogenic chloride secretion, induced by the second messenger cAMP, is the major driving force for intestinal epithelial cell ion secretion (2). Chemokine receptors are G protein-coupled receptors that transduce signals through pertussis toxin-sensitive Gi subunits (11, 35), which results in inhibition of adenylyl cyclases and cAMP production. Stimulation with CCL20, at concentrations that are produced by human intestinal epithelial cells (23), attenuated agonist-stimulated cAMP production and electrogenic chloride secretion by intestinal epithelial cells, and, consistent with signaling through G
i subunits, this inhibition was abrogated by pertussis toxin. Moreover, inhibition of agonist-stimulated cAMP production and its reversal by pertussis toxin was similar in CCL20- and somatostatin-treated cells, with the latter being a prototypic agonist that activates G
i subunits and inhibits adenylyl cyclases and cAMP (47, 48).
cAMP-dependent chloride secretion in colonic epithelial cells is dependent on cytoskeletal remodeling (17, 32, 43). Stabilization of F-actin with phalloidin, which prevents actin depolymerization, markedly inhibits forskolin-stimulated activity of the Na+-K+-2Cl cotransporter (NKCCl), a key component of the chloride secretory pathway (32, 33). Although we have shown that CCL20 partially inhibits forskolin-stimulated chloride secretion, doubtless due at least in part to a reduction in cAMP production, a possible influence of CCL20 on the cytoskeletal component of forskolin-stimulated ion transport should not be ruled out. Indeed, CCL20 stimulation of human colon epithelial cells resulted in tyrosine phosphorylation of p130Cas, an adaptor/scaffolding protein that associates with cytoskeletal and other focal adhesion proteins involved in cell adhesion and chemotaxis (6, 15, 37, 46). Cell migration requires the regulation of kinases and phosphatases, which modulate the phosphorylation and dephosphorylation of adaptor/scaffolding molecules in adhesions. Although little information is available regarding the activity of p130Cas in intestinal epithelial cells, in other cell types (e.g., fibroblasts) the FAK-Src complex mediates p130Cas tyrosine phosphorylation (49). Moreover, actin filament assembly and actin stress fiber organization is abnormal in p130Cas-deficient fibroblasts (19, 20). Our findings suggest the possibility that CCL20, in addition to its inhibitory effects on cAMP-dependent chloride secretion, has a role in epithelial cell adhesion/migration. If this is the case, it is tempting to speculate that alterations in intestinal epithelial cell adhesion/migration in response to CCL20 might also facilitate the passage of dendritic cell processes between epithelial cells (39).
Intestinal epithelial cells produce CCL20 and constitutively express its cognate receptor, CCR6 (23), which suggests that CCL20 can mediate autocrine/paracrine effects on intestinal epithelial cells. The gene encoding CCL20 is transcriptionally regulated by NF-B in human intestinal epithelial cells, and CCL20 is produced and secreted by those cells in response to proinflammatory mediators, microbial, infection and microbial products (e.g., bacterial flagellin) (23). The question then arises as to how, and under what conditions, CCL20 produced by intestinal epithelial cells alters intestinal epithelial cell functions. Our findings suggest that the answer to this question lies in the selective apical distribution of CCR6 on polarized intestinal epithelial cells. Stimulation of polarized intestinal epithelial cells with proinflammatory cytokines such as TNF-
or IL-1, which are produced by cells in the lamina propria and therefore stimulate the basolateral domains of epithelial cells, results in the basolateral secretion of CCL20 (23). Under those conditions, CCL20 would act mainly as a chemoattractant for target leukocyte populations in the lamina propria (e.g., dendritic cells, B cells) (7, 28). However, CCL20 access to the apical membrane could occur during inflammatory reactions or microbial infections sufficient to alter barrier integrity of the intestinal epithelium. In this case, CCL20 could signal epithelial cells through apical CCR6 receptors, leading to downregulation of epithelial secretory processes and changes in epithelial cell adhesion/migration that might favor restoration of the epithelial barrier. Alternatively, it should be considered that apically expressed CCR6 might serve as a target for ligands other than CCL20. For example, the antimicrobial peptide hBD2 is an NF-
B target gene that is upregulated in intestinal epithelium in response to proinflammatory signals and has been reported to signal leukocytes through CCR6 (36, 51). Consistent with the present data, we also have found CCR6 to be predominately apically expressed in human intestinal epithelium in vivo.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* C. C. Yang and H. Ogawa contributed equally to this work.
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