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Published online before print July 5, 2007
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,1
* Department of Clinical and Experimental Medicine, Hematology-Immunology Division,
Venetian Institute of Molecular Medicine, and
Department of Pathology, Padua University School of Medicine, Padova, Italy; and
Department of Pathology, Section of General Pathology, University of Verona, Verona, Italy
1 Correspondence: Padua University School of Medicine, Department of Clinical and Experimental Medicine, Hematology-Immunology Division, Via Giustiniani 2, 35128 Padova, Italy. E-mail: carlo.agostini{at}unipd.it
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In conclusion, T cells expressing CCR6, CXCR3, and CXCR6 act coordinately with respective ligands and Th1 inflammatory cytokines in the alveolitic/granuloma phases of the disease.
Key Words: T cells • macrophages • inflammation • lung
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cytokines and chemokines orchestrate the trafficking of immune-inflammatory cells to the lung during the development of the sarcoid process, favoring the entrance of effector cells and modulating local T cell survival and proliferation, into a Th1 pulmonary microenvironment [5
, 6
]. In particular, the interactions between chemokines and their receptors are believed to be responsible for the T cell recruitment during sarcoid hypersensitivity reactions. It has been demonstrated that at least two IFN--inducible CXC chemokines, CXCL10 and CXCL16, are crucially involved in Th1 cell migration into the lung of patients affected by sarcoidosis [7
, 8
]. CXCL10 and CXCL16, produced and released by macrophages and macrophage-derived cells, interact with their receptors CXCR3 and CXCR6, respectively, specifically coexpressed on the Th1 lymphocyte subset [8
, 9
]: The resulting intercellular signals affect the migration of sarcoid T cells into the lung and contribute to granuloma formation.
Another chemokine involved in trafficking and homing of lymphocytes into secondary lymphoid organs and in the maturation of leukocytes [10
] is the chemokine ligand 20, CCL20, also known as liver and activation-regulated chemokine [11
], Exodus-1 [12
], MIP-3
[13
], and small inducible cytokine subfamily A member 20 [14
], a molecule discovered through bioinformatic technologies. CCL20 stimulates the migration of B cells [15
], immature dendritic cells (DC) [16
], and a subset of memory T cells expressing the chemokine receptor CCR6 [17
]. CCL20 has a specific high affinity for CCR6 and is actually the only known chemokine ligand for CCR6 [18
]. CCL20 has been demonstrated in tonsillar crypts [19
], inflamed intestinal epithelial cells [20
], keratinocytes [21
], and in the lung, where it is inducibly expressed and secreted by the airway epithelial cells [22
, 23
].
In terms of the promigratory effect of CCL20 on T lymphocytes, we hypothesize that CCL20 may contribute to the development of the CD4+ T cell alveolitis, characterizing the initial phase of pulmonary sarcoidosis, and to the building of granulomatous structures. In particular, this study evaluated whether Th1 cells, accumulating in the lung of patients with sarcoidosis, express CCR6 and the eventual production and release of CCL20 by sarcoid alveolar macrophages (AMs). Using immunohistochemical studies, flow cytometry, and molecular analyses, we demonstrated that sarcoid CD4+ T lymphocytes obtained from the bronchoalveolar lavage (BAL) expressed CCR6. Moreover, we have shown that the CCR6/CCL20 interactions induce a migratory activity of sarcoid CD4+/CCR6+ T cells in vitro. Finally, we found that pulmonary macrophages, epithelioid cells, and epithelial cells, forming the central core of the granuloma, expressed and released detectable amounts of the chemokine CCL20.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Fifteen sarcoid patients presenting with an episode of pulmonary involvement were evaluated at the onset of the disease. They were defined as having an active disease on the basis of the following characteristics: lymphocytic alveolitis (>30x103 lymphocytes/ml); lung CD4:CD8 ratio >4.0; positivity to 67Gallium scan. The assessment of disease activity included BAL, clinical features, chest radiograph, lung function tests, high-resolution computed tomography, and routine blood studies.
BAL samples were also obtained from 18 patients with previously diagnosed pulmonary sarcoidosis, who repeated BAL fluid analysis during their follow-up period. These patients were in the chronic phase of the disease, as they had normal lung function, normal BAL fluid cell numbers, negative 67Gallium scan, and no clinical signs of acute disease. All patients were given steroid therapy previously (prednisone, 1 mg·kg–1·day–1), but no patient received immunosuppressive therapy for 6 months prior to the BAL analysis. The average follow-up period for this group of patients was 49 ± 11.2 months (range, 35–67 months).
Seven subjects were selected as controls for the BAL studies (three male and four female; mean age, 38.7±7.5 years; nonsmoker, evaluated for cough complaints without lung disease). They had a normal physical examination, chest X-rays, lung function tests, and BAL cell numbers.
Written, informed consent was obtained from each sarcoid patient and control subject.
Preparation of cell suspensions
Following administration of local anesthesia, BAL was performed as described previously [25
]. Briefly, a total of 150–200 ml saline solution was injected via fiber-optic bronchoscopy in 25 ml aliquots with immediate vacuum aspiration after each aliquot. The fluid was filtered through gauze, and its volume was measured. The amount of injected fluid recovered was 60.1 ± 8.9%. Cells recovered from the BAL fluid were washed three times with PBS, resuspended in endotoxin-tested RPMI 1640 (Sigma Chemical Co., St. Louis, MO, USA), supplemented with 20 mM HEPES and L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FCS (ICN Flow, Costa Mesa, CA, USA), and then counted. AMs, lymphocytes, neutrophils, and eosinophils were differentially counted in cytocentrifuged smears stained with Wright-Giemsa for a total count of 300 cells, according to morphological criteria.
AMs and BAL T cells were purified from the BAL cell suspensions by rosetting with neuraminidase-treated sheep RBCs followed by Ficoll-Hypaque gradient separations, as described previously. AMs were enriched further by removing residual CD3+, CD16+, and CD56+ lymphocytes with magnetic separation columns (MiniMACS, Miltenyi Biotec, Auburn, CA, USA), as described previously [25 ]. Staining with mAb showed that after this multistep selection procedure, >98% of AMs expressed the AM-associated CD68 antigen, whereas >98% of the rosetting population was constituted by CD3+ T cells.
PBMCs from the patients under study were obtained from freshly heparinized blood following centrifugation on a Ficoll-Hypaque gradient and washing with PBS. PBL were enriched further following rosetting of PBMC with sheep-RBCs, as reported above, and CD4+ T cells were separated from CD8+ T lymphocytes by magnetic separations over columns (MiniMACS). Briefly, the cell suspensions obtained as above were incubated at 4°C for 30 min with magnetic beads coated with anti-CD8 mAb (OKT8, Ortho, Raritan, NJ, USA). The CD8+ lymphocytes were then isolated and removed from CD4+ T cells by applying a magnetic system to the outer wall of the columns. After this selection procedure, >97% of the cells were viable, and 95–99% showed CD4+ phenotype.
mAb and cytokines
The commercially available, conjugated or unconjugated mAb used belonged to the Becton Dickinson/PharMingen (San Diego, CA, USA) series and included CD3, CD4, CD8, CD16, CD19, CD45RO, CD45RA, and isotype-matched controls. Anti-IL-4 and anti-IFN- mAb were purchased from PharMingen. Purified PE and FITC mouse anti-human CCR6, FITC anti-human CXCR3, purified anti-human CXCR6, and anti-human CCL20 mAb (R & D Systems Inc., Minneapolis, MN, USA) were also used.
The commercially available recombinant human IFN- (rhIFN-), rhTNF-, rhIL-2, rhIL-18, rhCCL20, rhCXCL10, and rhCXCL16 were purchased from R & D Systems Inc.
The frequency of BAL cells positive for the above reagents was determined by flow cytometry, as described previously [26 ]. Briefly, 10 µg/ml mAb were added to 0.5 x 106 cells, and the mixture was incubated for 30 min at +4°C. The cells were then washed twice and resuspended in 0.5 ml PBS for FACS analysis. For direct fluorescence analysis, FITC- or PE-conjugated control isotype-matched mouse mAb were used to set the fluorescence background (IgG1, IgG2a, and IgG2b, Becton Dickinson). For FACS analysis, 104 cells were acquired, and the expression was determined by overlaying the histograms of the samples stained with the different reagents. BAL lymphocytes and AMs were gated in flow cytometry analysis with two different approaches: physical characteristics of cells and expression of the T-associated CD3 and AM-associated pulmonary AM-1 antigens on the area of lymphocytes and AMs, respectively. The purity of the gates was always >98% of cells.
Cells were scored using a FACScan analyzer (Becton Dickinson), and data were processed with the Macintosh CELLQuest software program (Becton Dickinson). Expression of cytoplasmic cytokines was evaluated after fixing and permeabilization of the cell membranes with 100 µl Fix & Perm reagent (Caltag Laboratories, Burlingame, CA, USA) for 20 min. After these procedures, anti-IFN- and anti-IL-4 were added.
CCL20 protein levels in BAL fluid
CCL20 protein levels were measured in the fluid component of BAL recovered from 15 sarcoid patients (seven patients with active disease and eight with inactive disease). These experiments were performed using a specific double-determinant radioimmunoassay (RIA), as reported previously [27
]. Briefly, flat-bottomed, 96-well plates (MaxiSorp, Nunc, Roskilde, Denmark) were coated with 100 µl/well goat anti-human CCL20 antibody (R & D Systems Inc.; 5 µg/ml in 0.1 M carbonate buffer, pH 9.5) for 24 h at +4°C and then washed extensively with PBS, pH 7.5, 0.05 Tween 20 (washing buffer). CCL20 standards (Peprotech Inc., Rocky Hill, NJ, USA) or BAL fluids (100 µl/well) were then added, followed by an overnight incubation at +4°C. Plates were rinsed with washing buffer before addition of 100 µl 125I-labeled, affinity-purified, rabbit anti-human CCL20 polyclonal antibody (0.6 µg/ml in PBS-Tween with 50% FCS) and incubated overnight at 4°C. After washing the plates, 100 µl 1 N NaOH was added to each well, and samples were harvested after 30 min and read in a -counter. This RIA had a detection limit of 10 pg/ml.
In vitro production of CCL20 by pulmonary cells
To verify the ability of AMs to release the chemokine, unstimulated AMs (1x106/ml) were isolated from sarcoid patients, resuspended in RPMI medium, and cultured for 24 h in 24-well plates at 37° in 5% CO2. In separate experiments, AMs were stimulated with IFN- (100 U/ml), LPS (100 µg/ml), and TNF- (500 ng/ml; R & D Systems Inc.). Following the incubation period, the supernatants were harvested, filtered through a 0.45-µm Millipore filter, and stored immediately at –80°C. At the end of the culture time, AM viability was always >95%. CCL20 protein levels in supernatants were measured through the double-determinant RIA reported above.
Migration activity of pulmonary T cells in response to CCL20
T cell migration was measured in a 48-well, modified Boyden chamber (AC48, Neuro Probe Inc., Gaithersburg, MD, USA) made of two sections: Different chemotactic stimuli were loaded in the bottom section, and cells were added in the top compartment. Polyvinylpyrrolidone-free polycarbonate membranes with 5 µm pores (Osmonics, Livermore, CA, USA) and coated with fibronectin were placed between the two chamber parts. Only the bottom face of the filters was pretreated with fibronectin to increase the adherence of migrated cells. Before use, fibronectin-treated filters were washed extensively to avoid the shedding of fibronectin.
CCL20 (5.0 ng/ml), CXCL10 (100.0 ng/ml), and CXCL16 (120.0 ng/ml) chemokines were used to evaluate the migratory properties of pulmonary T lymphocytes from sarcoidosis patients. Chemokine or control medium (30 µl) was added to the bottom wells, and 50 µl T cells (5.0x106 cells/ml), resuspended in RPMI 1640, were added to the top wells. The chamber was incubated at 37°C with 5% CO2 for 2 h. The membranes were then removed, washed with PBS on the bottom side, fixed, and stained with DiffQuik (Dade AG, Düdingen, Switzerland). Cells were counted at 800x magnification in three fields per well. All assays were performed in triplicate.
CCL20 and CCR6 mRNA analysis
CCL20 and CCR6 mRNA levels were determined in the BAL cells recovered from 20 sarcoid patients (10 with active disease and 10 with inactive disease) and four control subjects. Total cellular RNA was extracted using the RNA Mini kit isolation and purification system (Qiagen Inc., Valencia, CA, USA) from 5–10 x 106-enriched BAL AMs or lymphocytes and quantified by measuring absorbance at 260 nm. cDNA was synthesized from 2 µg total RNA at 42°C for 15 min in the presence of avian myloblastosis virus RT (2.5 units), using 2.5 mM oligo-d(T) primer and reaction conditions described by the manufacturer (Promega Corp., Madison, WI, USA).
For CCR6, CCL20, and ß-actin mRNA detection, 1 µl cDNA was amplified in a volume of 50 µl in the presence of 25 pmol/l each specific oligonucleotide: 5'-ATC CTg CCA gAg CgA AAA gC and 3'-CAT TgT CgT TAT CTg Cgg TCT CAC, which give a band of 248 bp for CCR6; 5'-AAT CAg AAg CAg CAA gCA ACT and 3'-TTT TAC TgA ggA gAC gCA CAA, which give a band of 206 bp for CCL20; 5'-gTg ggg CgC CCC Agg CAC CA and 3'-CTC CTT AAT gTC ACg CAC gAT TTC, which give a band of 540 bp for ß-actin. PCR reaction mixtures consisted of 1.5 mmol/L MgCl2, 50 mmol/l KCl, 10 mmol/l Tris-HCl, 0.2 mmol/l concentrations each deoxynucleotide triphosphate, and 2.5 U Taq polymerase (Perkin Elmer, Norwalk, CT, USA). Reaction conditions were: 30 s melting at 94°C (for CCR6, CCL20, and ß-actin), 30 s annealing at 55°C (for CCR6 and ß-actin), or 51°C (for CCL20) and 40 s extension at 72°C for 25 cycles, followed by a final extension of 7 min at 72°C (for CCR6, CCL20, and ß-actin).
Each PCR product (10 µl) was electrophoresed in a 1.8% agarose gel in Tris-boric acid-EDTA 1x buffer. Gels were stained with ethidium bromide and photographed.
Immunohistochemical analysis of CCR6+ cells and CCL20-producing cells
Immunostaining for CCR6 and its ligand was performed using anti-CCR6 and anti-CCL20 antibodies (R & D Systems Inc.). Paraffin-embedded sections (4 µm-thick) were used for immunostaining using the standard avidin-biotin complex method (Vectastain ABC kit, Vector Laboratories, Burlingame, CA, USA). The reliability of both antibodies in paraffin sections was compared with cryostatic (frozen) lung sections of two patients with active sarcoidosis. Sections were deparaffinized in xylene (5 min for three times) and rehydrated through graded ethanol (twice for 5 min in 100% ethanol, 3 min in 95% ethanol, 3 min in 70% ethanol, and 5 min in distilled H2O). For the microwave antigen retrieval procedure, slides were placed in a 2-L glass beaker containing 0.01 mol/L citrate buffer, pH 5.9, and microwaved at 800 W for 5 min for three times before cooling and equilibration in PBS. To neutralize endogenous peroxidase activity, slides were pretreated with 3% hydrogen peroxide for 5 min. The slides were then incubated with primary antibodies for 1 h in a humidified chamber at 37°C (anti-mAb, 1:200). Immunoreactivity was detected using biotinylated secondary antibodies (1:1000 goat anti-mouse and 1:50 rabbit anti-goat dilution in PBS-BSA buffer), incubated for 45 min, followed by a 30-min incubation with avidin-peroxidase (1:200), and visualized by a 7-min incubation with the use of 0.1% 3,3'-diaminobenzidene tetrahydrochloride as the chromogen. Parallel control slides were prepared lacking primary antibody or lacking primary and secondary antibodies or stained with normal sera to control for background reactivity.
Effect of sarcoid cytokines on the expression of CCR6 and Th profile in peripheral T lymphocytes obtained from patients with sarcoidosis
To test the effects of cytokines, which are known to be released in the sarcoid microenvironment on the expression of CCR6, a time-course experiment was performed using highly purified, peripheral CD4+ T cells. CD4+ T lymphocytes were cultured in 24-well plates at the concentration of 2 x 106 cells/ml (Corning, Corning, NY, USA) in the presence of medium alone, rhIFN- (10 ng/ml), rhIL-2 (400 IU/ml), and rhIL-18 (100 ng/ml) for 2 h, 24 h, and 8 days at 37°C in a 5% CO2 atmosphere. Cells were collected at different time incubations and washed twice with PBS, and the frequency of T cells expressing CCR6 was determined by flow cytometry as described above.
Furthermore, CD4+ T lymphocytes were cultured in the presence of PHA (2.5 µg/ml) and the previous mentioned cytokines rhIL-2 and rhIL-18 for 24 h to evaluate the cytoplasmic IL-4 and IFN- cytokine expression of CCR6+ T cells as described above.
Statistical analysis
Data were analyzed with the assistance of the Statistical Analysis System. Data are expressed as mean ± SD. Mean values were compared using the ANOVA test. To investigate the correlation coefficients (r) between CCL20 levels and BAL cell findings, the nonparametric Spearman Rank correlation test was used. A P value <0.05 was considered significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(data not shown) [8
]. CD4+ and CD8+ T cell subsets and AMs detected in the BAL of patients with inactive sarcoidosis were superimposable to those observed in controls (Table 1)
.
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BAL CD4+ T lymphocytes obtained from patients with active sarcoidosis bore CCR6 [45.6±7.8% of CD4+ T cells, mean fluorescence intensity (MFI) average: 18.9±4.8; Fig. 1A and 1B ]. It is interesting that the expression of this receptor decreased significantly on BAL T cells obtained from patients with the chronic form of the disease (13.8±6.3% of CD4+ T cells; MFI average: 4.1±2.9; P<.05; Fig. 1C and 1D ) and from controls (12.1±4.5% of CD4+ T cells; MFI average: 3.9±3.2; P<.05; Fig. 1E and 1F ).
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at the cytoplasmic level. Independently by disease activity, remnant lung CD8+ T lymphocytes never expressed CCR6. In fact, pulmonary CD8+ T cells obtained from patients with active and chronic sarcoidosis were negative for the CCL20 receptor (data not shown).
At the same time, the expression of CCR6 by sarcoid AMs was never detected on the cell surface, and it was seen in the cytoplasm of the macrophages: in particular, AMs of patients with active sarcoidosis expressed high levels of the receptor (51.4±7.3% of AMs; MFI average: 139.5±43.8), and AMs obtained from patients with the inactive phase of the disease and from control subjects showed low levels or did not bear CCR6 (5.4±4.9% of AMs; MFI average: 7.3±5.3; 0% of AMs, respectively; data not shown).
CCL20 detection in BAL fluid component and BAL cells
Detectable levels of CCL20 protein were demonstrated in all BAL fluid components (range, 22–217 pg/ml BAL fluid component; Fig. 3
). CCL20 levels were significantly higher in BAL fluid components of patients with active sarcoidosis with respect to chronic patients and control subjects (190±39 pg/ml, 38±7 pg/ml, and 27±5 pg/ml, respectively; P<.01).
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Immunohistochemical analysis of CCR6 and CCL20 expression
Immunohistochemical analysis confirmed the high-intensity expression of CCR6 by sarcoid lung T cells infiltrating pulmonary biopsies obtained from three patients with active sarcoidosis (Fig. 4A
). Immunohistochemical analysis performed to investigate the cell sources of CCL20 in sarcoid tissue showed that the CCR6 ligand was preferentially expressed by macrophages, multinucleated giant cells, and epithelioid cells localized inside the granuloma (Fig. 4B)
. Immunohistochemical analysis of native lungs obtained from three patients with refractory sarcoidosis and pulmonary fibrosis who underwent lung allograft transplantation showed that in the fibrotic phase of the disease, lung T cells were mainly nonreactive for CCR6 (Fig. 4C)
, and AMs were not stained with anti-CCL20 mAb (Fig. 4D)
. It is interesting that some epithelial cells along the alveolar wall were reactive for the chemokine.
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Figure 5
shows the in vitro production of CCL20 by AMs. Cell-free supernatants were obtained from AMs cultured in different experimental conditions and tested for the presence of CCL20. After 24 h of culture, unstimulated AMs from sarcoid patients with active disease were able to produce higher levels of CCL20 (mean, 5311±307 pg/ml) than AMs isolated from patients with chronic disease (1499±298 pg/ml; P<.05) and control subjects (amount of CCL20 not detectable; P=<.01). Following LPS stimulation, AMs from patients with active and inactive sarcoidosis increased CCL20 release (7494±1870 pg/ml and 3807±1810 pg/ml, respectively), and control, LPS-stimulated AMs did not show any detectable release of CCL20 (P<.01). When AMs were cultured in the presence of IFN-, the amount of CCL20 in cell-free supernatants obtained from patients affected by active or chronic disease and from controls did not show significant differences with respect to unstimulated AMs (4856±402 pg/ml, 983±591 pg/ml, and nonquantifiable amount of CCL20, respectively; P=NS). TNF--cultured AMs, obtained from patients with active and inactive sarcoidosis, increased CCL20 release (7290±802 pg/ml and 3797±1001 pg/ml, respectively; P<.05); control TNF--stimulated AMs released smaller amounts of the chemokine (237±98 pg/ml; P<.01).
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(Table 2
and Fig. 7
). The intensity of CCR6 expression was evaluated by flow cytometry after incubating the cultures in the presence of the above cytokines for 2 h, 24 h, and after a long-term culture (8 days). CCR6 expression on peripheral T cells was increased after just 2 h of incubation with the above cytokines with respect to T lymphocytes cultured in medium alone. CCR6 expression persisted after 24 h and 8 days of culture (Table 2)
.
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induces the increase of CCR6 expression, we evaluated the effect of IL-2, IL-18, and PHA, a polyclonal mitogen, on IL-4 and IFN- production by peripheral blood CCR6+ T cells. The cytoplasmic cytokine expression was evaluated by flow cytometry after incubating the cells in the presence of the above cytokines for 24 h. After 24 h of culture, unstimulated peripheral blood CCR6+ T cells were IFN--negative and expressed cytoplasmic IL-4 (96.7±2.1%; MFI average: 35.5±8.4). Stimulated peripheral blood CCR6+ T lymphocytes showed the same cytokine profile with no significant changes (data not shown). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Pulmonary sarcoidosis is an interstitial lung disease characterized by a highly polarized Th1 immune response with specific Th1 cytokine (IFN-, IL-2, IL-12, IL-18) [5
, 28
] and chemokine (CXCL10, CXCL16, CCL5) [7
, 8
, 29
] profiles. CCL20 does not represent the typical Th1 chemokine: in fact, it is not stimulated by IFN- (as are CXCR3 and CXCR6 ligands), and data from literature report that in periodontal disease tissues, IFN- activity reduces gingival fibroblast CCL20 production induced by IL-1ß [30
]. Conversely, IFN- exerts a stimulating effect on CCR6 expression, i.e., a CCL20-specific receptor, on sarcoid T cells. At the same time, CCL20 production and release by airway epithelial cells [22
, 23
, 31
] and by sarcoid AMs (as reported in this study) can be induced strongly by TNF- and IL-1ß, two crucial effector cytokines of sarcoid Th1 immune response [22
].
AMs are the main source of CCL20 in the lung of patients affected by sarcoidosis. It is interesting that AMs did not bear CCR6 on cell surface, but we revealed the receptor at a cytoplasmic level. Chemokine receptors usually undergo a basal level of internalization, even in the absence of the specific ligand(s), with a process rate depending on the cell type [32 ] and the internalization pathways [33 ]. Receptor internalization is an essential mechanism for regulating leukocyte traffic and at the same time, for rendering a cell unresponsive to chemokines. In fact, the binding to the specific ligand(s) can enhance the internalization of the receptors greatly, although with different efficiencies affected by the type of ligand [34 , 35 ]. From this point of view, CCR6 exclusive cytoplasmic presence in AMs of patients might be ascribed to their considerable CCL20 production, which saturates CCR6 bonds, induces its surface leaving and causes CCR6 internalization. Unfortunately, we have no data to understand the dynamics of the receptor degradation or recycling and how CCR6 fate may affect the length and the strength of intracellular signals generated in AMs.
CCL20 is indeed overexpressed in a wide variety of diseases as a direct consequence of the stimulatory effects of other chemokines, such as IL-4, IL-13 [23
], and IL-17 [36
], in addition to TNF- and IL-1ß. The disorders mentioned above range from allergic pulmonary inflammation [37
, 38
], characterized by a Th2-polarized environment, to autoimmune encephalomyelitis [39
], to disorders with typical localized Th1 polarization, such as Crohn's disease [20
, 40
] and psoriasis vulgaris [41
]. In this context, the up-regulation of CCL20 in the lung of patients affected by sarcoidosis might be a consequence of local inflammation (associated with increased pulmonary levels of sarcoid cytokines) rather than an underlying cause. In fact, CCL20 is controlled directly by the transcriptional factor NF-
B, which is in turn up-regulated by TNF- [42
] and IL-1ß [43
].
The increased levels of CCL20 chemokine in the sarcoid pulmonary microenvironment amplify the immune-inflammatory response, originally elicited by an unknown antigen. As demonstrated by experiments established in CCR6–/– mice, the CCL20/CCR6 axis has a central role in chemoattracting specific T cell and DC subsets from the blood within the lung [31 , 44 ] and in the development of the pulmonary T lymphocyte activation [45 ]. Evidence from the literature and from our results suggests that in the sarcoid lung, immature CCR6+ DC, specialized for antigen uptake, become functional APC in response to inflammatory stimuli and trigger T cell responce. At the same time, CCR6+ T lymphocytes, recruited by CCL20, accumulate in the sarcoid pulmonary microenvironment, attending to the progression of the immune-inflammatory alveolitis and granuloma formation. Our immunohistologic studies localized CCR6+ T cells in the area surrounding the granuloma and showed the presence of CCL20+ cells in the core of the structure, indicating that CCR6/CCL20 interactions are involved in the assembly of the granuloma, in addition to CXCR3/CXCL10 and CXCR6/CXCL16 axes [8 ]. It is interesting that Qiu and colleagues [46 ], evaluating 24 different chemokine transcripts in mouse lungs, demonstrated the association between the increased CCL20 mRNA expression levels and the granuloma formation during the early phase of mycobacterial, antigen-elicited granuloma (a Th1 cytokine-associated granuloma) organization and the late phase of schistosomal antigen-elicited granuloma (a Th2 cytokine-associated granuloma) formation, underlining that CCL20 up-regulation is tied more strongly to inflammatory reactions rather than a Th-1- or -2-polarized environment.
Recently, a new subset of IL-10-producing, regulatory/effector T cells (Treg) was defined on the basis of the coexpression markers CD4, CD25, and CD45RO and the chemokine receptor CCR6 [47
]. As this lymphocyte subset most likely controls the immune response directly at the inflamed sites, it might be considered a natural counterbalance to the effector/memory T lymphocytes acting at the sites of ongoing inflammation. It is interesting, but not surprising, that we did not detect the CCR6+CD25+CD4+ Treg population in the lung of our patients with active or chronic sarcoidosis (data not shown): in fact, the memory CCR6+CD45RO+CD4+ pulmonary T cells, recruited by CCL20, never coexpressed the IL-2R subunit, i.e., CD25 marker [26
].
Despite the large amount of epidemiologic and immunohistologic studies, the etiology of sarcoidosis remains uncertain, even if it has emerged that the responsible agent(s) has to induce a Th1-polarized, immune response [48 ]. It is noteworthy that some chemokines, up-regulated in the sarcoid lung, showed antimicrobial activity as a further host-defense function of these molecules: CXCR3 ligands (CXCL9, CXCL10, and CXCL11) [49 ], CXCL16 [50 ], and CCL20 [51 ] exhibit the capacity to kill microorganisms in vitro. In particular, CCL20 released in the lung [22 ] displays antimicrobial activity, mainly against Gram-negative bacteria, and permeabilizes bacterial membranes such as human ß-defensin (HBD) [52 ]: HBD-1 and HBD-2 are two cationic, antimicrobial peptides involved in innate immunity, which share a high degree of structural homology with CCL20 and have also been shown to stimulate memory T cells and immature DC via a CCR6 receptor [53 ].
The chemokine axes CXCR3/CXCL10, CXCR6/CXCL16, and CCR6/CCL20 are involved in the recruitment and accumulation of leukocytes in the lung of patients with sarcoidosis. At the same time, the expression of distinct chemokine receptors on infiltrating T lymphocytes provides a potentially attractive opportunity to attenuate the influx of these cells in the lung. In fact, CCR6 expressed on immature DC has been identified recently as a therapeutic target in allograft rejections [54
, 55
]. Furthermore, in patients with psoriasis, the use of soluble TNF receptors induced the decrease of CXCL10 and CCL20 chemokines, which probably account for the reduced infiltration of T cells and DC in psoriatic plaques [56
]. It is interesting that some cases of sarcoidosis, resistant to conventional therapy, have been treated successfully with anti-TNF- [57
58
59
]. Therefore, a combined blockade of chemokine receptors and TNF- might be considered as a potentially promising approach in the therapy for sarcoidosis.
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ACKNOWLEDGEMENTS |
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Received March 2, 2007; revised June 8, 2007; accepted June 10, 2007.
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