Epithelial Inflammation Is Associated with CCL28 Production and the Recruitment of Regulatory T Cells Expressing CCR10

Bertus Eksteen, Alice Miles, Stuart M. Curbishley, Chris Tselepis, Allister J. Grant, Lucy S. K. Walker and David H. Adams
J Immunol July 1, 2006, 177 (1) 593-603; DOI: https://doi.org/10.4049/jimmunol.177.1.593

Abstract

Mucosal tissues require constant immune surveillance to clear harmful pathogens while maintaining tolerance to self Ags. Regulatory T cells (Tregs) play a central role in this process and expression of αEβ7 has been reported to define a subset of Tregs with tropism for inflamed tissues. However, the signals responsible for recruiting Tregs to epithelial surfaces are poorly understood. We have isolated a subset of CCR10-expressing CD25+CD4+Foxp3+ Tregs with potent anti-inflammatory properties from chronically inflamed human liver. The CCR10+ Tregs were detected around bile ducts that expressed increased levels of the CCR10 ligand CCL28. CCL28 was secreted by primary human cholangiocytes in vitro in response to LPS, IL-1β, or bile acids. Exposure of CCR10+ Tregs to CCL28 in vitro stimulated migration and adhesion to mucosal addressin cell adhesion molecule-1 and VCAM-1. Liver-derived CCR10+ Tregs expressed low levels of CCR7 but high levels of CXCR3, a chemokine receptor associated with infiltration into inflamed tissue and contained a subset of αEβ7+ cells. We propose that CXCR3 promotes the recruitment of Tregs to inflamed tissues and CCR10 allows them to respond to CCL28 secreted by epithelial cells resulting in the accumulation of CCR10+ Tregs at mucosal surfaces.

Chronic inflammation leads to tissue damage and the release of multiple potential autoantigens. Although secondary immune responses against such Ags can be detected, they are not a dominant feature of most chronic inflammatory diseases suggesting that mechanisms exist to suppress the adaptive immune response in inflamed tissues. Regulatory T cells (Tregs)3 have evolved to limit the local damage resulting from infectious challenges to the host. Natural Tregs arise in the thymus and survive as well as operate in the periphery by responding to a large variety of self-Ags (1, 2, 3). Tregs are CD4 cells that have a distinct phenotype characterized by expression of the a subunit of the high affinity IL-2R, CD25, and the glucocorticoid-induced TNFR (4). Despite displaying diverse TCR, there is evidence to suggest Tregs have a higher propensity to recognize self peptides than conventional CD25− T cells (3, 5). The most specific Treg marker is the transcription factor Foxp3, which is critical for Treg function. Retroviral transfer of Foxp3 to naive T cells converts them into functional Tregs, whereas its deletion ablates regulatory function and triggers autoimmunity (6, 7). Tregs also constitutively express the negative regulatory receptor CTLA-4, which binds the ligands CD80/CD86 and may be an important determinant of Treg function because CTLA-4-deficient mice have a similar phenotype to Foxp3 deficiency (8, 7). A recent study suggests that Tregs do not express the inhibitory receptor programmed cell death-1 (PD-1) on their surface, although it is retained intracellularly. This finding discriminates them from CD4+/CD25+ effector cells that express high levels of cell surface PD-1 (9).

Tregs control inflammation by contact-dependent TGF-β and IL-10 production (10, 11) and are able to control experimental gut inflammation in adoptive transfer models (12). Paradoxically, Tregs are required to maintain chronic intestinal inflammation in animal models, presumably by dampening more aggressive acute inflammation (13). Epithelial surfaces in particular are vulnerable to invasion by microbes and are a frequent target of chronic inflammatory diseases (14). Although Tregs have been reported in inflamed peripheral tissues, little is known about their function or the homing mechanisms that localize them to epithelial sites (15).

The chemokine receptor CCR10 is detected on both T and B lymphocytes at epithelial sites (16, 17) and defines subsets of lymphocytes that can be recruited to either mucosal or cutaneous epithelial sites. Mucosal homing is driven by the ligand CCL28, which is expressed by columnar epithelia in the gut, lung, breast and salivary glands (18), whereas homing to the skin is triggered by the alternative CCR10 ligand, CCL27 (19). Specificity is enhanced by the coexpression of CCR10 with organ-specific adhesion receptors. Thus mucosal CCR10+ lymphocytes also express α4β7 required for recruitment to the gut, whereas CCR10+ lymphocytes, which show tropism for the skin, coexpress the skin-homing receptor CCR4 and the cutaneous lymphocyte Ag (19).

CCL28 is constitutively expressed in the colon and increased by proinflammatory cytokines (20) and bacterial products, suggesting it has a role in recruiting effector cells to areas of epithelial injury (21). CCR10 expression has also been reported on nonlymphoid malignant cells (22), and coexpression of CCR9 and CCR10 has been implicated in the formation of small bowel melanoma metastases (23). CCR10+ lymphocytes are positioned in the intraepithelial compartment where coexpression of αEβ7 integrins (24) allows them to interact with E-cadherin expressed at epithelial adherens junctions (25). The expression of αE integrins also defines a population of CD25+ Tregs with enhanced suppressive properties compared with αE−CD25+ Tregs (26). Recent studies have suggested that the function of αE+ Tregs is at least in part dependent on their ability to be recruited to inflamed tissue compartments (27). Experimental animals that lack fucosultransferase required to synthesize E- and P-selectin ligands during inflammation are unable to recruit αE+ effector/memory Tregs to inflammatory sites and as a consequence display a reduced ability to suppress inflammation (27). In contrast, naive Tregs (CD25+αE−) express CCR7 and CD62L, allowing them to enter lymphoid tissues to control naive CD4 cell proliferation during the early phases of inflammation. We hypothesized that the chemokine receptor CCR10 would define a population of Tregs associated with epithelial inflammation. We used chronic liver disease as a model of persistent epithelial inflammation to test the hypothesis in humans. Inflammatory and autoimmune liver damage is often focused on the biliary epithelium, which is contiguous with the mucosa of the gastrointestinal tract and in primary sclerosing cholangitis (PSC) immune damage to bile ducts is mediated by mucosal T cells recruited as a consequence of aberrant expression of the gut homing molecules CCL25 and mucosal addressin cell adhesion molecule-1 (MAdCAM-1) (28, 29).

We report that CCL28 is expressed by biliary epithelial cells (BEC) in PSC and other forms of chronic liver disease (primary biliary cirrhosis (PBC), alcoholic liver disease (ALD)) with evidence of biliary inflammation. Expression of CCL28 by BEC is stimulated by LPS and IL-1β and recruits subsets of T lymphocytes expressing CCR10, which include CD4+CD25+Foxp3+ Tregs with potent suppressive properties. Thus CCL28 may be a critical signal for positioning Tregs at inflamed epithelial surfaces and CCR10 expression defines a subset of Tregs that home to epithelial sites.

Materials and Methods

Tissues

Diseased liver tissue and paired peripheral blood was obtained at the time of liver transplantation. Skin tissue was obtained from tissue removed during reconstructive plastic surgery. All samples were collected with appropriate patient consent and local research ethics committee approval.

Abs and reagents

The following primary Abs were used: mouse anti-human CCL28 mAb (62705, 5 μg/ml; R&D Systems), mouse anti-human α4β7 (ACT-1, 15 μg/ml; a gift from M. Briskin (Millennium Pharmaceuticals, Cambridge, MA), CD31 mAb (JC70A, 1 μg/ml; DakoCytomation), cytokeratin-19 mAb (BA17, 1 μg/ml; DakoCytomation), MAdCAM-1 mAb (CA 102.2C1, 12.5 μg/ml; Serotec), mouse anti-human anti-β1 mAb (3S3, 4 μg/ml; Abcam), mouse anti-human mAb HEA-125 (Progen), mouse anti-human β7-Cy5 mAb (FIB504, 1/20; BD Pharmingen), goat anti-human CCR10 (ab3904, 0.05 μg/ml; Abcam), CD8-FITC mAb (DK25; 1/50; DakoCytomation), CD4-PE-Cy5 mAb (C7069; 1/50; DakoCytomation), CD3 R-PE-Cy7 mAb (UCHT1, 1/20; Beckman Coulter), CD25-FITC (F0801, 1/50; DakoCytomation), CD56-PE mAb (MOC-1, 1/30; DakoCytomation), CD104-FITC mAb (Ber-ACT8, 1/25; DakoCytomation), CD49d-FITC mAb (P4G9, 1/50; DakoCytomation), CCR7 mAb (fab197p, 1/20; R&D Systems), and CXCR3 mAb (fab160p, 1/20; R&D Systems). Relevant secondary Abs used were donkey anti-goat PE/FITC (1/100; Abcam), goat anti-mouse FITC (20 μg/ml; Southern Biotechnology Associates) and goat anti-mouse IgG1 or IgG2a Texas Red (20 μg/ml; Southern Biotechnology Associates).

Liver-derived lymphocyte isolation

Liver tissues were reduced to 5-mm3 cubes and homogenized at 230 rpm for 5 min in a Stomacher 400 circulator (Seward). For detection of Tregs, liver tissue was digested for another 5 min with collagenase type IV (Sigma-Aldrich). Homogenized tissues were filtered through a fine mesh and lymphocytes were separated using 33%/77% (v/v) Percoll (Amersham Biosciences) density gradient centrifugation at 2000 rpm (650 × g) for 30 min.

PBL isolation

Lymphocytes were isolated from peripheral venous blood and diluted 1/1 with PBS before centrifuged over a Lymphoprep (Invitrogen Life Technologies) gradient for 30 min at 2000 rpm (650 × g).

Isolation and culture of BEC

BEC were isolated according to previously described methods (30). Briefly, liver tissue was finely chopped and subjected to enzyme digestion (collagenase type IV) and density gradient centrifugation. Nonparenchymal cells were then removed and separated by immunomagnetic selection. Cells positive for mAb HEA-125 (Progen) were classified as BEC. Following isolation BEC were plated in collagen coated flasks (Corning). BEC were cultured in DMEM, Ham’s F12, containing 10% human serum, penicillin and streptomycin (100 IU/ml), glutamine (2 mM), epidermal growth factor (10 ng/ml), hydrocortisone (2 mg/ml), choleratoxin (10 ng/ml), triiodo-thyronine (2 nM), insulin (0.124 U/ml), hepatocyte growth factor (5 ng/ml), and vascular endothelial growth factor (10 ng/ml). Cells were cultured to confluence and in all experiments used between passage 3 and 4.

In vitro BEC stimulation

BEC from six livers (n = 3 PSC, n = 1 PBC, n = 2 ALD) were grown to confluence in 24-well plates and either left under basal conditions or stimulated with cytokines for 24 h rTNF-α, IL-1β, IFN-γ (all 10 ng/ml from PeproTech), LPS (1 μg/ml), or 500 μM CDCA (chenodeoxycholic acid; Sigma-Aldrich). Following stimulation, mRNA as well as the supernatant was isolated from each well and stored at −70°C until analyzed. All treatments were performed in triplicate for each experiment.

Immunohistochemistry and dual color coimmunofluorescence

Six cases each of PSC, normal liver, PBC, and ALD and three normal spleens were examined. Sections for immunohistochemistry were initially incubated with 20% normal goat serum for 1 h before the addition of a mouse anti-human CCL28 mAb for 60 min. Control sections were incubated without the primary Ab or mouse Igs. Subsequently, sections were incubated sequentially for 20 min with biotinylated secondary Abs followed by a HRP complex (StepABCcomplex Duet kit; DakoCytomation). Sections were incubated with diaminobenzidine as a peroxidase substrate for 5–10 min until a color reaction developed and counterstained with hematoxylin (Mayer’s hemalum). Positive CCL28 staining was identified by the presence of a dark brown reaction product. All washes were performed with Tris-buffered saline (pH 7.6). Paraffin-fixed sections were initially dewaxed in xylene for 10 min and dehydrated in 100% ethanol for 10 min. Sections were washed and microwave Ag retrieval was done for 10 min in 0.01 M trisodium citrate buffer (pH 6). Sections were subsequently blocked, stained, and counterstained as per frozen sections described.

Sections for dual immunofluorescence were incubated with 20% normal goat/mouse serum for 30 min before the addition of primary Abs raised against CCR10 or CCL28 for 1 h. Control sections were incubated without primary Ab. Subsequently, sections were incubated with either CD31-Texas Red, HEA-labeled with Texas Red, or Foxp3-FITC and goat anti-mouse FITC (20 μg/ml) or rabbit anti-goat Texas Red (20 μg/ml) secondary Abs for 60 min in the dark. Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole. Dual immunofluorescence was assessed using AxioVision software.

Western blotting

Frozen tissue samples (n = 6 PSC, n = 6 PBC, n = 6 normal liver, n = 6 ALD, and n = 3 spleen) were homogenized into loading sample buffer and normalized for total protein content using Coomassie blue staining of gels or β-actin expression. Samples of equal protein content were loaded on 8% SDS-PAGE gels and following electrophoresis were transferred onto Hybond membranes (Amersham Biosciences). The membranes were blocked with 10% skim milk and subsequently incubated with mouse anti-human CCL28 Abs (2.5 μg/ml) for 1 h. The blots were washed and incubated with a goat anti-mouse HRP-conjugated Ab for 45 min. Immunoreactive bands were detected using the ECL detection system (Amersham Pharmacia). Bands were scanned on a Gel Doc (Bio-Rad) system and optical densities for individual bands compared.

Real-time RT-PCR for CCL28

RNA was extracted from snap frozen tissue (n = 6 PSC, n = 6 PBC, n = 6 normal liver, n = 6 ALD, n = 3 spleen, n = 3 skin) or stimulated BEC cultures (n = 3 PSC, n = 1 PBC, n = 2 ALD) using an RNeasy Mini kit (Qiagen). mRNA were transcribed to cDNA and real-time PCR performed on a PE7700 ABI Prism machine. Each reaction was performed in triplicate using QuantiTect Probe RT-PCR kit (Qiagen) according to the manufacturer’s instructions. Reactions contained 400 nM CCL28-specific 5′-CAGAGAGGACTCGCCATCGT and 3′-TGTGAAACCTCCGTGCAACA primers (AltaBioscience) and 200 nM CCL28-specific TaqMan probe 5′-FAM-CATGCCTCAGAAGCCATACTTCCCATTG-TAMRA-3′ (Eurogentech). Data are presented as fold increases in gene expression (ΔCT) normalized to the 18 S control and compared with normal skin tissue or untreated BEC (normalized to 1). Samples with no cDNA were used to control for background fluorescent signals.

RT-PCR for Foxp, CCR10, and IL-10

RNA was extracted from snap frozen tissue lymphocytes using an RNeasy Mini kit (Qiagen). Foxp3 and CCR10 mRNA were reverse transcribed and PCR performed using Qiagen One-Step RT-PCR kit according to manufacturer’s instructions. PCR products were normalized to β-actin mRNA expression and separated on a 1% agarose gel containing ethidium bromide (Sigma-Aldrich). Positive bands were detected under UV light and quantified by densitometry (Gel Doc; Bio-Rad). Primers were designed from GenBank sequences for CCR10 and Foxp3: CCR10 forward 5′-GAGGCCACAGAGCAGGTTTC-3′, reverse 5′-CATCGGCCTTGTAGCAAAGC-3′; and Foxp3 forward 5′-ACACCACCCACCACCGCC ACT-3′, reverse 5′-TCGGATGATGCCACAGATGAAGC-3′. IL-10, β-actin, and GAPDH primer pairs were obtained from R&D Systems.

CCL28 sandwich ELISA

The concentration of CCL28 in BEC supernatant samples (n = 6) were determined by sandwich ELISA (31) using human CCL28 DuoSet kits (DY717; R&D Systems). Briefly, relevant capture Abs were prepared in 0.1 M carbonate/bicarbonate buffer (pH 9.6), added to 96-well immunosorp plates (Nunc) and allowed to coat overnight at 4°C. Protein standards of known concentration or test samples were then added to relevant wells and incubated at room temperature for 1 h before washing and further 1 h incubation with relevant biotinylated detection Abs. The plates were then washed thoroughly before incubation with peroxidase-conjugated streptavidin (1/5000; Zymed Laboratories). The ELISA was developed using TMB substrate (3,5,3,5-tetramethylbenzidine, 100 μg/ml) in 0.11 M sodium acetate (pH 5.5), and 0.0003% H2O2 (both from BDH) and the enzymatic reaction was stopped using 2.5 M H2SO4. Colorimetric analysis was performed by measuring absorbance values at 450 nm. All measurements were performed using duplicate samples for each experiment.

Four-color flow cytometry

Lymphocytes were initially incubated with 3 mg/ml mouse Igs before incubation with a CCR10 Ab (0.05 μg/ml) for 30 min. Cells were washed, centrifuged for 10 min at 2000 rpm (650 × g), and labeled with a donkey anti-goat FITC/R-PE secondary Ab. Cells were subsequently washed and labeled with fluorochrome-labeled primary mAbs. Control samples were labeled with matched isotope control Ig. Samples were run on a Coulter Epics XL flow cytometer. Results were analyzed using Summit software (DakoCytomation).

Lymphocyte enrichment and culture

Lymphocytes were positively enriched to 95% purity for CCR10 with EasySep (StemCell Technologies). Briefly, lymphocytes were suspended in cold PBS with 1 mM EDTA and 2% FCS. Fc receptors were blocked (100 μl/ml EasySep human Fc blocker) for 10 min and followed by incubation with CCR10 R-PE primary Ab for 15 min. Lymphocytes were centrifuged at 10 min at 2000 rpm (650 × g) and subsequently incubated with EasySep FITC-PE selection mix, followed by magnetic nanoparticles. Magnetic positive selection of labeled cells was then performed and the purity of selected populations was confirmed by flow cytometry.

Tregs were isolated using the CD4+CD25+ Treg kit (Dynal Biotech) according to the manufacturer’s instructions. A total of 1 × 108 lymphocytes was suspended in 1 ml of PBS (pH 7.4) with 0.1% BSA and negative isolation of CD4+ cells performed by magnetic depletion with Abs to CD14, CD56, CD19, CD8, and CD235a. CD25+ regulatory cells were subsequently positively isolated by CD25 Abs conjugated with Dynabeads. Beads were removed from the cells by DETACHaBEAD (Dynal Biotech). Subsequent enrichment of Tregs based on CCR10 expression was done as described. Purity of isolated populations was confirmed by flow cytometry and Treg status confirmed by Foxp3 RT-PCR.

Lymphocyte stimulation, IL-10 capture, and IL-10 detection by FACS

Enriched populations of CD4+ lymphocytes were labeled with an IL-10 capture construct to retain secreted IL-10 (IL-10 secretion assay; Miltenyi Biotec) and stimulated for 4 h at 37°C in the presence of 50 ng/ml PHA or 1000 IU IL-2 (proleukin). Lymphocytes were subsequently washed, labeled with mouse anti-human IL-10 PE mAb (Miltenyi Biotec) and IL-10 production detected by flow cytometry. Unstimulated lymphocytes and isotype-matched control Abs were used as controls for flow cytometry.

Allogeneic lymphocyte proliferation assay

Liver lymphocytes expressing CD4 were isolated by negative selection and divided into CD25+CD4+ and CD25−CD4+ subsets by MACS using CD25 mAb and Dynabeads (Dynal Biotech). Beads were detached and removed with DETACHaBEAD (Dynal Biotech). CCR10+ populations were subsequently isolated using the described EasySep techniques. Matched naive PBL were labeled with CFSE (Sigma-Aldrich). CD25+CD4+ or CD25−CD4+ lymphocytes were cultured 1:1 with allogenic myeloid dendritic cells (isolated as previously described) (32) and CFSE-labeled cells added in ratios 1:1 to 1:30. Samples were cultured for 7 days and cell division measured by flow cytometry. For some experiments the CD25+CD4+ populations were further isolated based on αE integrin expression and repeated experiments as described.

Proliferation was also measured by incorporation of [3H]thymidine. CD25+CD4+ or CD25−CD4+ lymphocytes were cultured with autologous naive lymphocytes in ratios 1:1 to 1:30 with added 5 μl/ml anti-human CD3/CD28 beads (Dynal Biotech). After 24 h, 0.5 μCi [3H]thymidine was added to each well and the plate harvested on day 3. Results of radioactive thymidine are reported in cpm.

Transwell chemotaxis of lymphocytes

The migration of lymphocytes from inflamed livers (n = 4) and peripheral blood (n = 4) were assessed using fibronectin-covered (Sigma-Aldrich) 6.5-mm diameter, 5-μm pore Transwell inserts (Corning). Responses to CXCL12 were positive controls because large numbers of liver-infiltrating lymphocytes (LIL) express CXCR4. The 100 ng/ml recombinant human CCL28 or 100 ng/ml recombinant human CXCL12 was placed in the bottom of the well, and 5 × 105 lymphocytes were added to the upper chamber. Cells were collected from the top and bottom chambers after 2 h and measured by fixed volume counting and phenotyped for CCR10 expression by flow cytometry. Assays were conducted in duplicate and compared with control wells that only contained medium with BSA alone. Results of migrated cells are reported as a percentage of input cells.

Static adhesion assay

Isolated LIL from inflamed liver (n = 6) were phenotyped by flow cytometry for CCR10 expression and subsequently used in the static adhesion assay. Briefly, 18-well Teflon-coated slides (Erie Scientific) were incubated with recombinant human VCAM-1 (10 μg/ml), recombinant MAdCAM-1 (10 μg/ml) or BSA (1 μg/ml) at 4°C for 16 h in a humidified chamber. Slides were blocked with 10% FCS for 60 min before the addition of lymphocytes. Binding of lymphocytes to MAdCAM-1 or VCAM-1 was blocked by adding an anti-human α4β7 Ab or anti-β1 Ab, respectively, for 30 min at 37°C before lymphocytes were added. A total of 8 × 104 cells was added per well and allowed to bind for 10 min at 37°C. Some lymphocytes were preincubated with pertussis toxins (100 ng/ml). Adhesion was triggered by the addition of CCL28 (10 ng/ml; PeproTech) or MnCl2 (100 nM/ml). After incubation nonadherent cells were washed away using cold PBS. Slides were analyzed by manual counting of adherent lymphocytes in three representative high-power fields (hpf) per well.

Statistical analysis

Paired or independent t tests were used to assess data normally distributed, whereas nonparametric data were compared using Wilcoxon signed rank test (for related samples) or Mann-Whitney U test (for unrelated samples). Statistical analyses were performed with Axum 7 software.

Results

Increased expression of CCL28 in chronic liver disease

The chronic biliary diseases, PSC and PBC, are characterized by a chronic, predominantly T cell infiltrate in the portal tracts that is centered on inflamed bile ducts (Fig. 1⇓a). We investigated whether the epithelial chemokine CCL28 might be involved in this recruitment by staining frozen liver tissue from patients with chronic inflammatory liver disease with Abs against CCL28 (Fig. 1⇓b). Normal liver and splenic tissue revealed little or no detectable CCL28, whereas CCL28 was readily observed in the portal tracts of patients with PSC, PBC, and ALD. CCL28 expression was particularly intense on the cell membranes of injured bile ducts but was also detected on portal endothelium and on reactive bile ductules. Western blot analysis from whole liver protein lysates confirmed little CCL28 expression in normal liver and spleen tissue but a significant increase in CCL28 in inflamed liver tissue (Fig. 1⇓c). Total protein loading of samples was normalized by β-actin staining. PSC and PBC consistently expressed the highest levels of CCL28. mRNA from the same liver samples was analyzed by real-time RT-PCR using CCL28-specific primers and TaqMan probes (Fig. 1⇓d). Skin was used as a negative control and results normalized to 18S mRNA expression. Normal liver tissue contained 4-fold higher levels of CCL28 mRNA when compared with skin (p = 0.006) and chronically inflamed liver tissue showed a 15- to 25-fold increase in CCL28 mRNA with the highest levels again seen in PSC and PBC (p < 0.001). Selected tissues (PSC sample shown) were stained by dual immunofluorescence using the anti-CD28 mAb and Abs against either an endothelial marker (CD31) or an epithelial Ag (HEA-125). The results confirmed the previous cellular distribution of staining with intense coexpression of CCL28 and CD31 on portal vascular endothelium and with HEA-125 on biliary epithelium. Lower levels of CCL28 were detectable on sinusoidal endothelium. The distribution of CCL28 on BEC was consistent with membranous expression (Fig. 2⇓).

FIGURE 1.
FIGURE 1.

Livers from patients with chronic inflammatory liver diseases, including PSC, PBC, and ALD are heavily infiltrated by CD3+ lymphocytes. a, T cells are centered on inflamed bile ducts (arrow). Immunohistochemistry revealed intense CCL28 staining (detected as a brown pigment) on inflamed bile ducts (B) and to a lesser extent on portal vein (PV) and hepatic artery (A) endothelium. b, Little staining was seen on normal liver or spleen. Control sections had no detectable staining. We confirmed our observations with Western blotting (c), which demonstrated minimal CCL28 protein in normal liver and spleen but enhanced expression in chronic liver disease. Sample loading was normalized for β-actin and band intensity quantified using a Gel Doc system. Real-time RT-PCR of total liver mRNA samples confirmed increased CCL28 mRNA in diseased liver (p < 0.001, Student’s t test) but also normal liver (p = 0.006, Student t test) compared with skin after correction for 18S mRNA levels. d, Samples consisted of tissue samples from six donors each for PSC, PBC, normal liver, and ALD and three donors of spleen and skin tissue samples. Representative immunohistochemistry sections and Western blot results are shown. Results are expressed as mean ± SEM. ∗, p = 0.006 ∗∗, p < 0.001.

FIGURE 2.
FIGURE 2.

Coimmunofluorescence was used to confirm the cellular distribution of CCL28. Abs to CCL28 labeled with FITC green colocalized with anti-CD31 mAb labeled with Texas Red (TxRED) on portal endothelium (upper; yellow) product. There was little CCL28 expression on sinusoidal endothelium (middle). The strongest staining of CCL28 was detected on bile ducts and where anti-CCL28 labeled with FITC colocalized with an Ab against the epithelial Ag HEA labeled with Texas Red (lower). The staining shown on tissue sections from a patient with PSC. The findings are representative of the patterns of immunofluorescence staining seen in tissue sections from six donors each for PSC, PBC, and ALD. Sections stained with control Abs demonstrated minimal background tissue fluorescence.

IL-1 and LPS stimulate CCL28 release by BEC

To determine the factors that stimulate CCL28 secretion by epithelial cells, primary human BEC (cholangiocytes) were isolated from liver tissue removed at transplantation using established techniques and grown to confluence (Fig. 3⇓a). Phenotype was confirmed by cytokeratin-19 expression and all experiments were done with cells at passage 4 or less. Cholangiocytes were grown in 24-well plates and either left under basal conditions or stimulated with the following factors for 24 h: recombinant TNF-α, IFN-γ, TNF-α with IFN-γ, and IL-1β (all 10 ng/ml), LPS (1 μg/ml), or 500 μM of the bile acid CDCA. CCL28 protein was measured using ELISA (Fig. 3⇓b) and mRNA with real-time PCR (Fig. 3⇓c). Stimulation with TNF-α, IFN-γ alone, or a combination of both did not induce expression of CCL28, whereas both IL-1β and LPS induced high levels of CCL28 mRNA and secretion of protein (p < 0.002). CDCA induced CCL28 expression to a lesser but still significant extent (p < 0.01).

FIGURE 3.
FIGURE 3.

To determine factors that induce CCL28 expression, we isolated and cultured primary BEC from human livers (a) and stimulated confluent highly pure cultures for 24 h with combinations of TNF-α, IFN-γ, TNF-α, IL-1β (all 10 ng/ml), LPS (1 μg/ml), or 500 μM of the bile acid CDCA. CCL28 protein was measured by sandwich ELISA (b) and mRNA by real-time PCR (c). LPS and IL-1β were the most potent inducers of CCL28 expression (∗, p < 0.002). Treatment with CDCA also induced CCL28 secretion (∗∗, p < 0.01), but at a lower level than LPS and IL-1β treatment. Experiments reflect BEC isolated from six donors (n = 3 PSC, n = 1 PBC, n = 2 ALD) and assays conducted in triplicate. Results are expressed as mean ± SEM. ∗∗, p < 0.01 ∗, p < 0.002 by Student t test compared with unstimulated cultures.

CCR10+ CD4 T cells are enriched in inflamed liver tissue

Given the identification of CCL28 in inflamed liver, we hypothesized that a fraction of the lymphocytes within the inflammatory lesion might have been recruited on the basis of CCR10 expression. Lymphocytes were therefore isolated from the livers of patients with chronic liver disease (samples, n = 6 PSC, n = 6 PBC, and n = 6 ALD) and their phenotype compared with that of PBL using flow cytometry (Fig. 4⇓a). Gut-derived CD19+ B cells were used as a positive control. This analysis revealed that CCR10 was expressed by 16.7 ± 4.1% of CD3+ T cells derived from diseased liver tissue. The majority of CCR10 expression on intrahepatic T cells was confined to the CD4 compartment with only low levels of CCR10 detectable on CD8+ T cells and CD56+ NK cells (33). The patterns of expression of CCR10 obtained by flow cytometry were confirmed using RT-PCR to assess levels of CCR10 mRNA in subpopulations of lymphocytes from the liver. Consistent with the flow cytometry data, the CCR10 message was largely restricted to the CD3+ and CD4+ subsets (Fig. 4⇓b). Trace amounts of CCR10 mRNA within the CD56+ and CD8+ populations may reflect contamination with CD4+ cells as magnetic separation only selects to 95% purity.

FIGURE 4.
FIGURE 4.

CCR10 expression on LIL. a, CCR10 expression by CD3+ T cells derived from diseased liver tissue were assessed by flow cytometry and compared with isotype-matched negative control samples. Gut CD19+ B cells were used as a positive control. CCR10 was expressed by 11.4 ± 3.0% (n = 18) of the LIL with most staining seen on CD4+ T cells and little staining of CD56+ NK cells and CD8+ T cells. As shown, CCR10 is expressed by 7.6% of CD4+ T cells. Parentheses show value for the mean channel fluorescence. b, CCR10 expression by the CD4+ subset was confirmed using RT-PCR with primers specific for human CCR10 mRNA on subsets of T cells purified by immunomagnetic selection as described in Materials and Methods. A representative agarose gel is shown; overall samples from 10 donors were assayed in duplicate. c, There were significant differences in the expression of CCR10 on CD4 T cells derived from different tissues. A representative experiment of CCR10 expression on gated CD4 T cells (left). Isotype-matched control (open histogram) and representative samples (grayed overlay histograms) are shown for peripheral blood (0.4%), normal liver (6.2%), and inflamed liver (15.6%). Overall 2.2 ± 1.8% (n = 10) of PBL CD4+ T cells were CCR10+, 6.3 ± 3.5% (n = 6) of CD4+ T cells in normal liver expressed CCR10 (∗, p < 0.04 compared with PBL), and 11.4 ± 3.0% (n = 18) of liver-infiltrating CD4 T cells in chronic inflammatory liver disease (∗, p < 0.04 compared with normal liver or ∗∗, p = 0.002 compared with PBL). d, The majority of CCR10+CD4+ cells expressed α4β1 integrins (78.6 ± 5.1%), whereas subpopulations expressed α4β7 (11.1 ± 4.4%) and αEβ7 (3.5 ± 2.0%) integrins. Representative flow cytometry histograms are shown from 10 PBL, six normal liver, and 18 inflamed liver (n = 6 PSC, n = 6 PBC, and n = 6 ALD) donors. Results are expressed as the mean ± SEM. Values for p were determined by Student’s t test.

We extended detection of CCR10 on CD4+ T cells to include lymphocytes derived from peripheral blood and from normal liver tissue. CCR10 was expressed at lower frequencies on PBL and normal liver CD4+ T cells compared with T cells isolated from inflamed liver (Fig. 4⇑c); 2.2 ± 1.8% of CD4+ T cells in blood expressed CCR10 compared with 6.3 ± 3.5% of CD4 T cells isolated from noninflamed liver (p < 0.04 compared with blood). CCR10+CD4+ T cells were further enriched in inflamed liver tissue with 11.4 ± 3.0% CD4+ T cells expressing CCR10 (p < 0.04 compared with normal liver or p < 0.002 compared with PBL).

Because integrins act in conjunction with chemokines to regulate the homing of T cells, we analyzed integrin expression on the liver-infiltrating CCR10+CD4+ T cells (Fig. 4⇑d). Virtually all CCR10+CD4+ T cells expressed α4β1 integrin (78.6 ± 5.1%), whereas subpopulations expressed α4β7 (11.1 ± 4.4%) and αEβ7 (3.5 ± 2.0%) integrins. Results are expressed as the mean ± SEM and were obtained following subtraction of control samples that were stained with isotype-matched control Abs.

CCL28 stimulates CCR10-dependent migration and β1 and β7 integrin activation on liver-derived lymphocytes

Our finding of α4, β1, and β7 integrins on CCR10+ lymphocytes in the liver led us to determine whether CCL28 can trigger integrin-mediated adhesion of LIL to immobilized MAdCAM-1 and VCAM-1, both of which are present on portal endothelium in chronically inflamed liver. Exposure of LIL to 10 ng/ml CCL28 activated integrin-mediated adhesion to both recombinant MAd-CAM-1 (78 ± 2.8 cells/hpf) and recombinant VCAM-1 (240 ± 13.4 cells/hpf) (both p < 0.02 compared with BSA controls). Adhesion was inhibited by preincubation of lymphocytes with pertussis toxin, suggesting G protein-coupled receptors were required, and blocking Abs to α4β7 reduced binding to MAdCAM-1, whereas anti-β1 Abs inhibited adhesion to VCAM-1. Manganese was used as a positive control as a nonselective activator of integrin adhesion cells without CCL28 to determine basal binding and BSA as a control substrate (Fig. 5⇓a). Thus CCL28 binding to CCR10 can activate both β1 and β7 integrins.

FIGURE 5.
FIGURE 5.

LIL express functional CCR10. a, The ability of CCL28 to activate lymphocyte binding to MAdCAM-1 and VCAM-1 was studied using total T lymphocyte populations from diseased livers (n = 6). Treatment of LIL with either 10 ng or 50 ng/ml CCL28 significantly increased adhesion to both VCAM-1 and MAdCAM-1. Adhesion was inhibited by preincubation of lymphocytes with pertussis toxin (ptx) or blocking mAbs to either β1 or α4β7 integrins. Manganese (Mn) was used as a positive control as a nonsignaling activator of integrin adhesion. b, Migration of LIL to CCL28 was assessed using fibronectin-coated Transwell migration chambers. CXCL12 and BSA were use as controls. A total of 5 × 105 lymphocytes isolated from inflamed livers (n = 4) and peripheral blood (n = 4) were loaded per well, and experiments were done in duplicate. Liver lymphocytes showed significant migration to CCL28 compared with BSA (∗, p < 0.004). Phenotyping by flow cytometry of the lymphocytes that migrated to CCL28 revealed that the migrated lymphocytes were predominantly CCR10+ and include both CCR10+ T cells (45 ± 3.1%) and CCR10+ B cells (33.4 ± 2.2%). The majority of migrated T cells were CD4+ and included both CD25+ and CD25− subsets. Representative FACS plots with percentage expression are shown. Results are expressed as mean ± SEM. ∗, p < 0.004 and ∗∗, p < 0.03 by Student’s t test and Wilcoxon signed rank test where appropriate.

Transwell migration assays were used to demonstrate that CCL28 can promote chemotaxis of CCR10+ lymphocytes isolated from inflamed human livers. BSA was used as a negative control and 100 ng/ml CXCL12 as a positive control (Fig. 5⇑b). PBL (11.1 ± 3.7%; p < 0.03) and LIL (16.0 ± 3.9%; p < 0.004) demonstrated significant migration to CCL28 compared with BSA controls. Phenotyping by flow cytometry of the lymphocytes that migrated to CCL28 revealed that the vast majority were T cells expressing CCR10 (45 ± 3.1%) with a smaller population of B cells (33.4 ± 2.2%). The majority of migrated T cells were CD4+ and included both CD25+ and CD25− subsets. Thus, CCR10 expressed on liver infiltrating CD4+ lymphocytes support migration to CCL28.

LIL contain a population of Foxp3+ Tregs that express CCR10

Given the role of Tregs in maintaining chronic inflammation in animal models (34), we investigated whether lymphocytes in inflamed human livers contain a population of Tregs that express CCR10. Using flow cytometry and negative CD4+ T cell enrichment, we were able to identify a population of CD4+CD25+ T cells (10.2 ± 4.8%) within the total CD4 population of the inflamed liver. Gating on CCR10+ expression demonstrated that 42.6% of CD25+CD4+ T cells expressed CCR10. To confirm that the CCR10-expressing lymphocytes in inflamed liver include a population of Tregs, we used RT-PCR to analyze the Treg-specific transcription factor Foxp3. mRNA for Foxp3 was clearly identified within lymphocytes isolated from the inflamed liver. Moreover, when we used immunomagnetic isolation to enrich or deplete for CCR10, Foxp3 mRNA was preferentially located in the CCR10-enriched fraction. This finding implies that the T cell infiltrate in inflamed liver tissue includes a population of Foxp3+ Tregs that express CCR10 (Fig. 6⇓a).

FIGURE 6.
FIGURE 6.

Characterization of CCR10 expression by Tregs in human liver. a, CD4+ T cells were enriched from inflamed livers and flow cytometry was used to identify the CD25+CD4+ population. These cells comprised 10.2 ± 4.8% of the total CD4+ T cell infiltrate. Gating on CCR10+ expression revealed that 42.6% of the CCR10+ cells were CD25+CD4+ T cells. b, To confirm that the CCR10+ T cell population in inflamed liver includes a population of bona fide Tregs, rather than activated CD25+ T cells, we performed RT-PCR for the Treg-specific transcription factor Foxp3. mRNA for Foxp3 was clearly identified within total lymphocyte isolates from the inflamed liver. The expression of Foxp3 on CCR10+ liver T cells was determined by magnetic sorting of lymphocytes isolated from inflamed liver tissue to obtain CCR10+ and CCR10− populations followed by RT-PCR with specific human Foxp3 primers. Intense expression of Foxp3 was detected in the CCR10-enriched population and confirmed the presence of Tregs within the CCR10+ population. Results are expressed as mean ± SEM. Representative FACS plots with percentage expression and agarose gels are shown (n = 6). c, Immunohistochemistry was used to investigate the tissue location of Foxp3+ Tregs. In the normal liver Foxp3+ Tregs were infrequent but were consistently located in close proximity to bile ducts (arrow). Foxp3+ Treg numbers were increased in chronic inflammatory liver disease predominantly in expanded, inflamed portal tracts. Dual immunofluorescence (bottom) using Abs to Foxp3-Texas Red and CCR10 FITC-green revealed coexpression (yellow) of CCR10 on these portal Foxp3+ Tregs. Typical tissue sections are shown that were representative of the staining seen in tissue sections from six donors each for PSC, PBC, normal liver, and ALD.

To determine the tissue anatomical distribution of Foxp3 cells in the liver we conducted immunohistochemical analysis of liver tissues. In the normal liver Tregs occurred infrequently and were almost exclusively found in the portal tracts close to a bile duct (Fig. 6⇑c). There was a marked increase in the number of Foxp3+ cells in chronic inflammation with the majority detected in expanded and heavily inflamed portal tracts. Dual immunofluorescence confirmed coexpression (Fig. 6⇑c, lower panel, yellow) of Foxp3 in a subpopulation of the CCR10+ lymphocytes.

CCR10+ Foxp3+ intrahepatic Tregs are functional suppressor cells

We used allogeneic stimulation/suppression assays to confirm that CCR10+CD25+CD4+ lymphocytes are fully functional Tregs (Fig. 7⇓, a and b). Freshly isolated and unstimulated CD25+CD4+ cells were isolated from inflamed livers and enriched for CCR10 by positive immunomagnetic selection. Isolated lymphocyte fractions were cultured 1:1 with allogeneic myeloid dendritic cells and CFSE-labeled lymphocytes added in ratios 1:1 to 1:30. CD25−CD4+CCR10+ lymphocytes did not inhibit proliferation of stimulated CFSE-labeled naive T cells, whereas CD25+CD4+CCR10+ lymphocytes mediated potent, dose-dependent inhibition of proliferation (p < 0.003 compared with CD25− samples). Further enrichment for αE expression increased the suppressor activity, but this was not statistically significant. We confirmed the suppressive effects of CD25+CD4+CCR10+ T cells in a standard [3H]thymidine proliferation assay. Ratios of 1:5 to 1:1 with autologous naive T cells activated by anti-CD3/CD28 beads significantly reduced proliferation of naive lymphocytes (Fig. 7⇓c).

FIGURE 7.
FIGURE 7.

CCR10+ Tregs in human liver can suppress allogeneic proliferation in vitro. a and b, CD4+ liver lymphocytes were isolated from diseased liver tissue by negative selection and positively enriched for CD25 and CCR10 expression. Where indicated, CCR10+CD25+CD4+ Tregs were further isolated based on αE expression. Matched naive PBL were labeled with CFSE. CCR10+CD25+CD4+ or CCR10+CD25−CD4+ lymphocytes were cultured with allogeneic myeloid dendritic cells and with CFSE-labeled cells added in ratios 1:1 to 1:30. Samples were cultured for 7 days and cell division measured by flow cytometry. The mean number of cell divisions was calculated based on the number of CFSE+ cells per cell division and divided by the total viable cell count. CCR10+CD25+CD4+ Tregs inhibited proliferation compared with CD25− subsets (∗, p < 0.003; n = 4) at ratios of 1:1 to 1:10. CCR10+CD25+CD4+ αE+ populations appeared more potent at inhibiting proliferation, although the difference did not reach statistical significance (p = 0.09). Representative histograms from one of the flow cytometric analyses are shown. c, To further illustrate the suppressive effects of CCR10+CD25+CD4+ T cells, we used [3H]thymidine proliferation assays to confirm suppression of proliferation at 1:5 to 1:1 ratios with naive lymphocytes activated by anti-CD3/CD28 beads. Values for p were calculated by paired Student’s t test. d and e, IL-10 secretion by CD4+CD25+ Tregs isolated from liver tissue was demonstrated using an extracellular capture assay. CD4+CD25+ cells, further separated into CCR10− and CCR10+ subsets, were stimulated for 4 h with 1000 U/ml recombinant human IL-2, and expression of IL-10 was measured by extracellular capture detected by flow cytometry. Consistent with a regulatory phenotype, stimulation of CCR10+CD4+CD25+ cells in culture with PHA or by high dose IL-2 resulted in detectable IL-10 in 47 ± 4.4% and 42 ± 3.0% of the CCR10+CD4+CD25+ cells, respectively. Transcription of IL-10 by CD25+CD4+ and CD25+CD4+CCR10+ populations was confirmed by RT-PCR with GAPDH expression used as a comparator for loading (mean ± SEM; n = 4).

IL-10 production by CCR10+CD4+CD25+ intrahepatic T cells

A feature of Tregs is their ability to secrete the anti-inflammatory cytokine IL-10. To assess whether CD25+CD4+CCR10+ lymphocytes are able to produce IL-10, cell surface labeling was used to capture and detect IL-10 secreted by the different subpopulations of lymphocytes. CD4+CD25+ and CD4+CD25− subsets as well as those enriched for CCR10 expression were cultured with IL-2 or PHA and IL-10 detected by flow cytometry after 4 h. CD4+CD25+ and CD4+CD25+CCR10+ populations secreted IL-10 and this result was confirmed using RT-PCR and IL-10-specific primers with GAPDH used as a loading control (Fig. 7⇑, d and e).

CCR10+ Foxp3+ intrahepatic Tregs are CCR7low and express high levels of the inflammatory chemokine receptor CXCR3

CCR10+ Tregs from inflamed livers expressed high levels of CXCR3 and relatively low levels of CCR7 consistent with an activated, tissue infiltrating phenotype. This finding is in contrast to CCR10− Tregs in blood that expressed low levels of CXCR3 and high levels of CCR7 consistent with peripheral naive-like Tregs and a proclivity to migrate to secondary lymphoid tissue. Given the lack of a reliable surface marker to identify Tregs within the CD4+CD25+ T cell population, we used RT-PCR to confirm Foxp3 mRNA expression in the CXCR3+ and CCR7+CD25+CD4+CCR10+ populations (Fig. 8⇓).

FIGURE 8.
FIGURE 8.

CCR10+ Tregs have a tissue infiltrating phenotype that is distinct from CCR10− naive Tregs. a, Flow cytometry revealed that CCR10+ Tregs from inflamed livers expressed high levels of CXCR3 and relatively low levels of CCR7 whereas CCR10− Tregs from blood expressed low levels of CXCR3 and high levels of CCR7. b, Given the lack of a reliable surface markers for Tregs we measured expression of FoxP3 in CCR7 and CXCR3+ subsets using RT-PCR with primers specific for human FoxP3 and controlled sample loading based on β-actin expression. RT-PCR confirmed Foxp3 mRNA expression in the CCR10+ or CCR10− Treg populations that have been further enriched based on CXCR3+ and CCR7+ expression (n = 4). Representative FACS plots and agarose gels are shown. Results are expressed as mean ± SEM.

Discussion

Natural Tregs suppress tissue damage that occurs as a consequence of inflammatory or antimicrobial immune responses (15). Tregs are positively selected in the thymus as a consequence of their high affinity for self Ags expressed on thymic epithelium and can be detected in blood, lymphoid tissues, and inflamed peripheral tissues. Their function in vivo relies on their ability to produce IL-10 and surface-bound TGF-β but it remains unclear exactly where Tregs operate. A subset of “naive” circulating Tregs has recently been reported in the peripheral circulation, which express the lymph node homing receptors CCR7 and CD62L, suggesting that they home to secondary lymphoid tissue where they may suppress the proliferation of T cells during early stages of immune activation (27, 35). In contrast a subset of highly differentiated Tregs, which express the αE integrin is found in inflamed peripheral tissue where they exert anti-inflammatory properties (27). Inflammation is markedly increased in the absence of such cells and it is likely that they act to maintain suppression of immune responses and particularly to limit the tissue damage resulting from attempts to control infectious agents. Recent studies suggest that chronic inflammation is the result of a balance between pro- and anti-inflammatory pathways in which Tregs promote stable chronic inflammation while preventing fulminant destructive inflammation (34).

The factors responsible for the recruitment of Tregs to peripheral tissues are not well understood. CCR4, CCR8, and CXCR3 have been reported on Tregs and could provide entry to dermal sites (CCR4, CCR8) (36) and areas of general inflammation (CXCR3) (37). In addition, CCR4 and CCR8 ligands may also contribute to Treg-dendritic cell interactions after tissue entry (38). However, specific signals that promote trafficking of Tregs to epithelial sites such as the gut, lung, and liver are not known. CXCR3 ligands are expressed on endothelium and in some cases epithelium of inflamed tissues and thus might aid Treg entry but are not tissue specific (39). Our finding of CCR10 expression on a population of liver infiltrating Tregs suggests that CCL28 might provide a mucosal signal to selectively recruit Tregs to epithelial surfaces. The coexpression of CCR10 with CXCR3 we describe would augment recruitment of Tregs at inflamed epithelial sites such as the liver where CXCR3 ligands are strongly expressed (39). We detected CCR10+ Tregs in the chronically inflamed liver in close proximity to intrahepatic bile ducts the epithelial cells of which showed strong expression of CCL28. These intrahepatic Tregs expressed high levels of CXCR3, which is known to be critical for the recruitment of effector T cells to the inflamed liver (39), but low levels of the lymph node homing chemokine receptor CCR7. A subset of the CCR10+ Tregs expressed high levels of the αE integrin, which defines Tregs with increased anti-inflammatory properties, and confers on them the ability to be retained at mucosal surfaces by binding to E-cadherin on epithelial cells. A higher proportion of intrahepatic T cells were Tregs in the diseased livers, suggesting that these cells are recruited as a consequence of chronic inflammation to limit tissue damage. However, ∼10% of intrahepatic T cells in noninflamed livers from organ donors were Tregs, suggesting that these cells may play a role in suppressing responses in the liver under physiological conditions. This outcome is consistent with a recent murine study suggesting that Tregs play a critical role in regulating the transition from hepatic immune tolerance to hepatic inflammation (40).

We found very few CCR10+ Tregs in the peripheral circulation. This finding is in contrast to CCR7+ Tregs, which can be detected at relatively high frequencies in blood and secondary lymphoid tissue (35). This implies different pathways of homing for these two Treg subsets. It has been suggested that CCR7+ naive Tregs operate in secondary lymphoid tissues to suppress early events during T cell activation including the expression of chemokine receptors required for tissue infiltration (41). CCR4, CCR5, and CCR8 have been implicated in the recruitment of Tregs to lymphoid tissues (42, 43) but as yet very little is known about the signals responsible for recruiting Tregs to peripheral tissues. Our data suggest that CXCR3 and CCR10 might be critical in this process and it is possible that activation of naive Tregs in lymph nodes leads to the differentiation of Tregs with a tissue-infiltrating phenotype characterized by expression of CCR10, CXCR3, and in some cells high levels of αE integrin. Thus naive-type and tissue-infiltrating Tregs may operate at different sites and at different stages of the immune response to suppress inflammation. The fact that a subset of skin-homing T cells express CCR10 and respond to the other CCR10 ligand, CCL27 in the skin suggests that CCR10 expression on Tregs would allow them to enter both dermal and mucosal sites depending on their coexpression of additional skin- or gut-specific homing molecules (19, 44). Expression of gut and skin adhesion molecules are, however, subject to significant plasticity and could be altered by interactions with local dendritic cells to facilitate cross-homing between the gut and the skin (45).

Tregs are likely to play a critical role in the pathogenesis of chronic liver disease. Hepatitis C infection is characterized by increased numbers of circulating CD4+ Tregs, depletion of which enhances Ag-specific CD8 effector responses (46). Increased intrahepatic CD4+ Tregs have also been reported in the liver of patients with hepatocellular carcinoma (47). In both cases Tregs are implicated in the disease pathogenesis by suppressing potentially beneficial T cell responses against virus or tumor, respectively. Regulation of liver inflammation is not only restricted to CD4+ Tregs because an intrahepatic population of CD8+ T cells with regulatory activity has been reported in chronic hepatitis C infection (48). In chronic inflammatory disease, the role of Tregs may be more complex. We studied two diseases targeted to bile ducts, PBC and PSC. Autoimmune and infectious etiologies have been proposed for both conditions but no definitive pathogenic mechanism has been reported for either disease (49, 50). Both diseases are characterized by chronic inflammation focused on the biliary epithelium and both show a slow progression to fibrosis and cirrhosis. It is possible that Tregs are initially recruited to limit the tissue damage associated with bile duct inflammation and subsequently maintain the stability of chronic inflammation, which characterizes both diseases. Support for this model is provided by evidence that Tregs play such a role in animal models of chronic T cell-mediated inflammation in the lung and inflammatory bowel disease (34, 51). Chronic Ag exposure in the lung mucosa promotes the recruitment of Tregs and the establishment of a local microenvironment in which secretion of the proinflammatory cytokines IL-2 and IFN-γ is reduced and levels of IL-10 and IL-5 increased. The intrahepatic T cells we describe secrete IL-10 and may not only suppress inflammatory damage but also promote the development of a fibrogenic microenvironment and progression to fibrosis and cirrhosis.

In summary we propose that expression of CCL28 by epithelial cells in response to microbial products or IL-1 provides a signal to localize CCR10+ Tregs at mucosal surfaces. This report is the first linking a specific chemokine receptor with recruitment of Tregs to epithelial surfaces and suggests that CCR10 expression defines a population of Tregs with the capacity to traffic to mucosal sites to limit autoimmunity and inflammation.

Acknowledgments

We thank Gary Reynolds for expert help with immunohistochemistry as well as Janine Youster and Jean Shaw for contribution in the collection and processing of the tissue samples. We acknowledge the advice of Dr. Vincent Lai in isolating dendritic cell subsets.

Disclosures

The authors have no financial conflict of interest.

Footnotes

  • 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.

  • ↵1 This work was supported by grants from The Medical Research Council and Core Charity, U.K.

  • ↵2 Address correspondence and reprint requests to Dr. David H. Adams, Liver Research Group, Medical Research Council Centre for Immune Regulation, 5th Floor Institute for Biomedical Research, University of Birmingham, Birmingham B15 2TH, U.K. E-mail address: d.h.adams{at}bham.ac.uk

  • ↵3 Abbreviations used in this paper: Treg, regulatory T cell; LIL, liver-infiltrating lymphocyte; PSC, primary sclerosing cholangitis; PBC, primary biliary cirrhosis; ALD, alcoholic liver disease; MAdCAM-1, mucosal addressin cell adhesion molecule-1; hpf, high-power field; BEC, biliary epithelial cell; CDCA, chenodeoxycholic acid.

  • Received October 14, 2005.
  • Accepted April 12, 2006.

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