CCL18 Is Expressed in Atopic and Mediates Skin Homing of Human Memory T Cells

This information is current as Claudia Günther, Concha Bello-Fernandez, Tamara Kopp, of October 2, 2021. Julia Kund, Nicole Carballido-Perrig, Sonja Hinteregger, Sandra Fassl, Christoph Schwärzler, Günther Lametschwandtner, Georg Stingl, Tilo Biedermann and José M. Carballido J Immunol 2005; 174:1723-1728; ; doi: 10.4049/jimmunol.174.3.1723 Downloaded from http://www.jimmunol.org/content/174/3/1723

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CCL18 Is Expressed in Atopic Dermatitis and Mediates Skin Homing of Human Memory T Cells

Claudia Gu¨nther,* Concha Bello-Fernandez,† Tamara Kopp,‡ Julia Kund,* Nicole Carballido-Perrig,* Sonja Hinteregger,* Sandra Fassl,* Christoph Schwa¨rzler,* Gu¨nther Lametschwandtner,* Georg Stingl,‡ Tilo Biedermann,1* and Jose´M. Carballido2*

CCL18 is a human secreted by and dendritic cells. The receptor for CCL18 is not yet known and the functions of this chemokine on immune cells are not fully elucidated. In this study, we describe that CCL18 is present in skin biopsies of atopic dermatitis (AD) patients but not in normal or psoriatic skin. CCL18 was specifically expressed by APCs in the dermis and by Langerhans and inflammatory dendritic epidermal cells in the epidermis. In addition, the serum levels of CCL18 and the percentages of CCL18-producing / and dendritic cells were significantly increased in AD patients ؉ compared with healthy controls. Furthermore, we demonstrate that CCL18 binds to CLA T cells in peripheral blood of AD Downloaded from patients and healthy individuals and induces migration of AD-derived memory T cells in vitro and in human skin-transplanted SCID mice. These findings highlight a unique role of CCL18 in AD and reveal a novel function of this chemokine mediating skin homing of a subpopulation of human memory T cells. The Journal of Immunology, 2005, 174: 1723–1728.

he regulation of leukocyte trafficking during homeostatic in chronic AD, IFN-␥ secreting Th cells are also present (11). The

and pathologic responses is achieved in great part by che- suggested to mediate the homing of Th cells into AD http://www.jimmunol.org/ T mokines. Chemokines comprise a large group of homol- skin include the CCR4 ligands CCL17 (12) and CCL22 (13) and ogous proteins that exert their biological effects by interactions the CCR10 ligand CCL27 (14). However, additional chemotactic with cell surface heptahelical G protein-coupled receptors (1). signals may be required for the specific recruitment of specialized CCL18, also designated (DC)3-derived CC chemo- T cell subpopulations (15). In this study, we demonstrate that kine 1, pulmonary and activation-regulated chemokine, alternative CCL18 contributes to the leukocytic infiltration associated with activation-associated CC chemokine 1, and MIP-4, is AD. We found, that AD patients have elevated levels of CCL18 in a human chemokine structurally related to CCL3 (2, 3). So far, serum and lesional skin and that CCL18 recruits human memory T CCL18 has no mouse equivalent and its receptor is not known. cells derived from AD patients to human skin in vivo.

CCL18 was shown to be expressed in germinal centers of tonsils by guest on October 2, 2021 Ϫ by dendritic cells and to attract mainly naive T cells, CD38 man- Materials and Methods tle zone B lymphocytes and DC (2, 4). The production of CCL18 Patients by APC is enhanced by Th2 like IL-4 and IL-13 and suppressed by IFN-␥ (5, 6). Accordingly, CCL18 has been impli- Blood samples were obtained with informed consent from Caucasian in- cated in hypersensitivity pneumonitis (7), vernal keratoconjuncti- dividuals, including 36 patients with AD and 28 nonatopic healthy con- trols. Clinical severity was documented independently and blindly by the vitis (8), and contact hypersensitivity (9). Atopic dermatitis (AD) clinicians using the eczema area and severity index (EASI) and the inves- is a Th2-mediated, chronic inflammatory skin disease character- tigators’ global assessment (IGA) (16). The patient population was distrib- ized by eczematous skin lesions (10). Histopathologically, skin uted according to IGA scores as follows: almost clear (n ϭ 3), mild disease lesions of AD reveal a mononuclear cell infiltrate consisting of (n ϭ 0), moderate disease (n ϭ 15), severe disease (n ϭ 10), and very ϩ severe disease (n ϭ 4). The patients did not receive any systemic or topical macrophages and CD4 Th cells. In acute lesions, these Th cells treatment with immunosuppressive drugs for at least 4 wk before collection mainly produce Th2 cytokines like IL-4, IL-5, and IL-13, whereas of blood samples and skin biopsies.

Purification and culture of cells † *Novartis Institutes for Biomedical Research, Vienna, Austria; Department of Im- PBMC from human blood were prepared by density gradient separation on munology, Vienna International Research Cooperation Center, Vienna, Austria; and ‡Department of Dermatology, Division of Immunology, Allergy and Infectious Dis- Ficoll-Paque Plus (Amersham Biosciences). The AD-derived T cell lines eases, Vienna Medical University, Vienna, Austria (NIT, SVT, DK2-JOT) and clones (98016T.02, 98016T.24), which ex- pressed cutaneous lymphocyte Ag (CLA), CCR4, and displayed a Th2/Th0 Received for publication June 14, 2004. Accepted for publication November secretion pattern following activation, were generated from le- 15, 2004. sional punch biopsies as previously described (17, 18). T cells were cul- The costs of publication of this article were defrayed in part by the payment of page tured in X-Vivo15 medium (BioWittaker) supplemented with recombinant charges. This article must therefore be hereby marked advertisement in accordance human IL-2 (20 ng/ml; Novartis Institutes for Biomedical Research with 18 U.S.C. Section 1734 solely to indicate this fact. (NIBR) Vienna, Austria). 1 Current address: Department of Dermatology, Eberhard Karls University, Tu¨bingen, Germany. Flow cytometry 2 Address correspondence and reprint requests to Dr. Jose´M. Carballido, Novartis Institutes for Biomedical Research, Brunnerstrasse 59, A-1235 Vienna, Austria. E- Cells were stained for 30 min on ice using FACS buffer (PBS containing mail address: [email protected] 2% FCS and 0.1% sodium azide). The following mAbs and corresponding 3 Abbreviations used in this paper: DC, dendritic cell; AD, atopic dermatitis; EASI, isotypes were used: CD3-PE, CD3-PerCP, CD4-PE, CD4-PerCP, CD8- eczema area and severity index; IGA, investigators‘ global assessment; AP, alkaline PerCP, CD14-FITC, CD19-FITC, CD20-FITC, CD62-PE, CD56-FITC, phosphatase. HLA-DR-CyChrome, CCR4-PE, CD54-PE, CLA-FITC, rat anti-CLA,

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 1724 CCL18 MEDIATES SKIN HOMING OF MEMORY T CELLS

mouse anti-rat IgM-PE (BD Pharmingen), CD3-allophycocyanin, CD3- 3) were stained with goat anti-CCL18 mAb (R&D Systems) or goat serum FITC, CD4-allophycocyanin, CD11a-PE, CD11c-allophycocyanin, CD19- and biotinylated horse anti-goat IgG (Vector Laboratories) followed by allophycocyanin (Caltag Laboratories), CCR3-PE, CCR5-PE, CCR7-PE, avidin-conjugated alkaline phosphatase (AP) and naphthol-AS phosphate/ CXCR3-PE, CXCR4, and CXCR6-Pe (R&D Systems). Intracellular cyto- Fast Red TR substrate (Sigma-Aldrich). CCL18-binding cells in AD sam- kine production by T cells was determined after5hofstimulation with ples were detected using CCL18-biotin followed by avidin-AP and Fast anti-CD3mAb (1 ␮g/ml; BD Pharmingen) and 2 ng/ml PMA (Sigma-Al- Red. The samples were costained with anti-CD3 mAb (BD Biosciences) or drich). Brefeldin A (10 ␮g/ml; Epicentre Technologies) was added for the anti-CCR4 mAb (Santa Cruz Biotechnology) followed by anti-mouse-Ig- last 2 h. Thereafter, cells were fixed with 2% formaldehyde (Sigma-Al- biotin (Vector Laboratories), avidin-AP, and Fast Blue (Sigma-Aldrich) drich), subsequently permeabilized with 0.5% saponin (Sigma-Aldrich), and counterstained with hematoxylin (Merck). For immunofluorescence and intracytoplasmic cytokine production was detected using allophyco- analysis, cryostat sections were stained with goat anti-CCL18 (R&D Sys- cyanin- or PE-conjugated mAbs specific for IL-4 or IFN-␥, respectively tems) and mouse anti-HLA-DR or anti-CD3 (BD Biosciences), anti-CD1a (BD Pharmingen). CCL22-Alexa647 was purchased from Dictagene. (Caltag Laboratories), anti-CD207 (Baxter) followed by rabbit anti-goat- CCL18 (PeproTech) was labeled in-house with Cy5 or biotin (Molecular Texas Red and horse anti-mouse-FITC (Vector Laboratories), respectively. Probes) according to the manufacturer’s instructions. A peptide of similar Slides were mounted with 4Ј,6Ј-diamidino-2-phenylindole) containing me- size (CH1 domain of the intracellular protein P300), that has no known cell dium (Vector Laboratories), coverslipped, and analyzed with a confocal surface receptor, was also labeled with Cy5 and used as staining control. scanning microscope (Zeiss) using the Zeiss LSM5 Image Browser. Chemokines were used for staining at a final concentration of 300 ng/ml. Analysis was performed on a FACSCalibur (BD Biosciences) using BD Statistical analysis CellQuest software. Data are presented as bars (indicating averages Ϯ SE), curves (indicating Ϯ Detection of intracellular CCL18 in monocyte/macrophages and averages SE), or box plots (indicating average, median, 25–75% per- centiles and SD). Statistical analysis was performed using unpaired Stu- DC after stimulation with IL-4 dent’s t test with the help of Origin 7.0 software (OriginLab). Clinical data

PBMC (5 ϫ 105 cells/ml) from AD patients (n ϭ 8) and controls (n ϭ 13) were tested for simple correlations by determining Pearson’s correlation Downloaded from were cultured for 48 h in the presence or absence of 10 ng/ml recombinant coefficient and uncorrected probability values with Systat 10 software human IL-4 (NIBR) and treated with brefeldin A (10 ␮g/ml; Sigma-Al- (SSI). Statistical evaluation of histologic samples was performed using Ј Ͻ drich) for the last 3 h. Monocyte/macrophages were recognized by CD14 Fisher s exact test (Systat 10 software). In all cases, p 0.05 was con- staining, and myeloid DC were identified as the CD11cϩ fraction of HLA- sidered significant. DRϩ, linϪ (CD3, CD14, CD19, CD20, CD56) PBMC. For intracellular staining of CCL18, surface-stained cells were fixed with 4% paraformal- Results dehyde (Sigma-Aldrich), permeabilized with 0.5% saponin (Sigma-Al- CCL18 is expressed in human AD skin http://www.jimmunol.org/ drich), and stained with goat anti-human CCL18 mAb (R&D Systems) followed by donkey anti-goat-PE (DPC Biermann). Goat serum (Sigma- AD is an inflammatory skin disease associated with cutaneous hy- Aldrich) was used as isotype control. perreactivity to allergens and high IL-4 production (10). Conse- quently, AD can provide a suitable environment for CCL18 up- ELISA regulation. To test this hypothesis, we performed an Serum CCL18 and CCL22 levels were determined using Duoset ELISA immunohistochemistry evaluation of CCL18 expression in skin (R&D Systems) according to the manufacturer’s instructions. samples of AD patients (n ϭ 10) in comparison to normal (n ϭ 3) Chemotaxis assay and psoriatic (n ϭ 3) skin. CCL18 expression was detected in all AD skin samples but was absent in the skin from healthy or pso- Chemokines (R&D Systems) or control medium (RPMI 1640 without phe- by guest on October 2, 2021 nol red with 0.5% BSA) were added in 30 ␮l to the lower wells of a 96-well riatic individuals (Fig. 1A). The differences in the expression of chemotaxis chamber (NeuroProbe). Calcein-AM (1 ␮M; Molecular CCL18 in those skin samples were significant as assessed by Fish- Probes)-labeled cells were seeded at 3 ϫ 106 cells/ml in the upper chamber er’s exact test ( p Ͻ 0.003). In AD skin, CCL18 was strongly and selected wells of the lower chamber (to obtain 100% migration value). expressed in the upper dermal vascular plexus, in close association ␮ The two chamber compartments were separated by a 3- m pore size poly- with the mononuclear cell infiltrates. Immunofluorescence staining carbonate filter (NeuroProbe). After a 2.5-h incubation at 37°C, filters were ϩ removed, the plate was spun down, and supernatants were discarded. Then, demonstrated that CCL18 expression was restricted to HLA-DR 20 ␮l of 0.4% Triton X-100 (Sigma-Aldrich) was added to each well. The APC (Fig. 1B). Particularly, all CCL18-expressing cells in the epi- degree of migration was quantified by fluorescence reading using a Victor dermis were CD1aϩ. Subsequent staining with Abs against CD207 II (Wallac, PerkinElmer Life Sciences) multiwell plate reader. (langerin) demonstrated that most of the CCL18-expressing cells In vivo migration assay in SCID-hu Skin mice were Langerhans cells whereas the remaining population consisted of inflammatory dendritic epidermal cells. Keratinocytes, fibro- Human skin pieces were obtained from the West Hungarian Tissue Bank, blasts, and CD3-positive T cells did not express CCL18 (Fig. 1B). Petz Alada´r County Hospital (Gyo¨r, Hungary) according to institutional and governmental ethical guidelines. SCID (C.B-17/GbmsTac-Prkdcscid Lystbg) mice (Taconic) were transplanted on their backs with two human CCL18-producing APC are increased in blood of AD patients skin pieces from the same donor as previously described (19). Chemokine- Our findings demonstrating a selective expression of CCL18 by induced skin homing of human T cells was performed using a modification APC in the skin of atopic individuals prompted us to investigate of the previously described method (18). AD skin-derived T cells were labeled with CFSE (1 ␮M in PBS for 5 min at room temperature; Molec- whether CCL18-producing APC are also enriched in the circula- ular Probes) and 5 ϫ 107 cells/mouse were injected i.v. into SCID mice tion of these patients. CCL18 expression by APC of AD patients transplanted with human skin (SCID-hu Skin mice). Immediately after, and healthy controls was assessed by intracellular immunofluores- CCL18 or CCL22 (300 ng in 30 ␮l PBS; R&D Systems) or PBS alone were cence staining following stimulation with IL-4. As shown in Fig. injected intradermally into human skin grafts. Each single mouse was in- 2A, the percentages of CCL18-producing monocyte/macrophages jected with one chemokine in one human skin graft and with another che- ϩ mokine or with PBS control in the other skin graft. After 24 h, skin grafts (CD14 ) in blood of AD patients were significantly higher were explanted, processed into single-cell suspensions, stained, and ana- (Ͼ5-fold, p Ͻ 0.003) than those observed in healthy individu- ϩ ϩ lyzed in the FACS by quantifying the percentages of CD3 , CFSE cells als. Similarly, the percentages of CD11cϩ-, HLA-DRϩ-, and present in the living (propidium iodide negative) cell fraction. Alterna- linϪ CCL18-producing DC from AD patients were also higher tively, parts of the human skin grafts were frozen for further analysis by Ͻ immunohistochemistry. (3-fold, p 0.001) than those of healthy donors (Fig. 2A). In both groups, CCL18-expressing cells were detected following Immunohistology stimulation with IL-4, suggesting that CCL18 was produced de Acetone-fixed 5-␮m cryostat sections of normal skin of healthy subjects novo and that the reactivity was not due to staining of prestored (n ϭ 3) and lesional skin from patients with AD (n ϭ 10) or psoriasis (n ϭ CCL18 in cellular compartments (data not shown). CCL18 The Journal of Immunology 1725 Downloaded from

FIGURE 2. CCL18-producing APC and serum levels of CCL18 are in- creased in AD patients. A, PBMC from AD patients (n ϭ 8) and controls (n ϭ 13) were stimulated with IL-4. The percentages of CCL18-producing ϩ

monocyte/macrophages in PBMC and CCL18-producing CD11c blood http://www.jimmunol.org/ DC are represented as box plots. The analysis shows significant differences between monocyte/macrophages of AD and control individuals (averages, 7.1 and 1.3%, respectively, p Ͻ 0.003) and between DC of AD and control individuals (averages, 5.6 and 1.9%, respectively, p Ͻ 0.001). B, CCL18 levels in serum from AD patients (n ϭ 36) are significantly higher than those of healthy control (n ϭ 28) subjects (averages, 34.9 and 10.7 ng/ml, respectively, p Ͻ 0.0003). C, Graphical distribution of serum levels of CCL18 among AD patients with different severity disease scores (IGA). D, Correlation between CCL18 and CCL22 serum levels in AD patients. F, FIGURE 1. CCL18 is expressed in dermis and epidermis of AD pa- Patients with almost clear to severe manifestations (r ϭ 0.6, p Ͻ 0.003). by guest on October 2, 2021 tients. A, Frozen sections of lesional biopsies from AD, psoriasis, or Patients with highest severity scores, represented by E, are excluded from healthy skin were stained for CCL18 expression. B, Double-immunofluo- the correlation analysis. rescence staining with indicated Abs performed on frozen sections of AD skin biopsies. Original magnification, ϫ200 (A) and ϫ400 (B).

sion (Fig. 2D, E). Thus, the selective increase of CCL18 expres- production was undetectable in the CD3ϩ T cell and CD19ϩ B sion during the development of AD and its subsequent decline in cell fractions of PBMC cultured for 48 h in the presence or patients with the highest clinical disease scores suggests a unique absence of IL-4 (data not shown). pattern of CCL18 regulation.

AD patients have higher CCL18 serum levels than normal CCL18 binds to memory Th cells in AD individuals The detection of the cell population targeted by CCL18 is ham- Serum values of CCL18 were 3-fold higher ( p Ͻ 0.0003) in AD pered by the fact that the CCL18 receptor is not known. To over- patients (34.9 Ϯ 5.3 ng/ml, mean Ϯ SE) than in healthy, non- come this problem, we generated Cy5- and biotin-labeled CCL18 allergic, age- and sex-matched controls (10.7 Ϯ 0.9 ng/ml, mean Ϯ without altering their biological properties (e.g., Cy5- and biotin- SE; Fig. 2B). When the atopic patients were grouped according to labeled CCL18 were as potent as unlabeled CCL18 to induce con- their disease stage (determined by the clinical scores IGA and centration-dependent migration of PBMC in vitro; data not EASI), we observed that the expression levels of CCL18 in mod- shown). Using these tools, we found CCL18 binding to a fraction erate and severe AD were higher than in mild disease manifesta- of CD3ϩ T cells infiltrating the skin of AD patients (Fig. 3A). tions (Fig. 2C). However, the serum levels of CCL18 decreased in CCL18-binding cells represented approximately one-half of the patients with very severe disease scores (Fig. 2C). CCR4ϩ cells. In addition, most of the CCL18-binding cells coex- Other chemokines associated with AD, such as CCL17, CCL22, pressed CCR4 (Fig. 3A). Since the inflammatory T cell infiltrate of CCL26, and CCL27, have been described to correlate with disease AD consists mostly of memory T cells (21), we investigated severity scores (12–14, 20). In our patient population, we also whether CCL18 would interact with memory T cells. Indeed, found a correlation between EASI and serum CCL22 levels (r ϭ 5–10% of peripheral blood memory CD3ϩCLAϩ T cells in AD 0.4, p Ͻ 0.05; data not shown). Furthermore, we found a strong patients bound to CCL18 (Fig. 3B). The frequencies in CCL18- correlation (r ϭ 0.6, p Ͻ 0.003, Fig. 2D, F) between CCL18 and binding cells were slightly increased in the peripheral CCL22 expression in patients with mild to severe disease. How- CD3ϩCLAϩCCR4ϩ T cell compartment. In all cases, there was a ever, patients with the highest CCL22 serum levels, who also had nonsignificant increase in the percentages of CCL18-binding T the highest disease scores, showed only moderate CCL18 expres- cells detected in the circulation of AD donors compared with 1726 CCL18 MEDIATES SKIN HOMING OF MEMORY T CELLS

healthy controls (Fig. 3B). The percentages of cells reacting to an irrelevant fluorescently labeled peptide (P300) of similar size as CCL18 were indistinguishable from the percentages obtained in the absence of labeled chemokine and consistently below 1%. In addition, CCL18 also bound to CLAϩ memory T cell lines and clones isolated from the skin of AD patients, independently of whether they displayed a Th2 or Th1/0 cytokine production pattern following stimulation (Fig. 3C). Similarly, fluorescently labeled CCL22 reacted positively with all skin-derived T cells tested, whereas the control peptide P300 did not elucidate any significant reactivity (Fig. 3C). Particularly, CCL11 did not compete with CCL18. This is important since CCL11 is a ligand for CCR3 and this receptor. Using specific mAbs, we demonstrated that CCL18- binding cells were CCR4ϩ, but CCR3Ϫ, further excluding a pu- tative binding of CCL18 to CCR3 (Fig. 3E).

CCL18 recruits memory Th cells to the skin The functional relevance of CCL18 binding to memory Th cells was assessed in in vitro and in vivo migration assays. In vitro, all Downloaded from AD-derived T cell lines and clones tested, migrated significantly, and in a dose-dependent manner in response to CCL18 and to CCL22. The extent of CCL18-induced migration was comparable to that induced by CCL22 (Fig. 4A). Both CCL18- and CCL22- induced migration could be blocked using specific neutralizing mAbs, demonstrating the specificity of the responses (Fig. 4A). The role of CCL18 in vivo was investigated using a modifica- http://www.jimmunol.org/ tion of the previously described SCID-hu Skin mouse model (18). AD-derived memory T cell lines and clones migrated significantly to human skin grafts transplanted onto SCID mice in response to CCL18 alone (Fig. 4, B and C) with magnitudes that were com- parable to that mediated by CCL22. Under these conditions, the migratory response induced by CCL11 was insignificant and sim- ilar to that mediated by the control PBS (Fig. 4C), which is in

agreement with the lack of CCR3 expression by these AD-derived by guest on October 2, 2021 T cells (Fig. 3E). The extent of CCL18-mediated T cell infiltration observed in the mechanistic SCID-hu Skin mouse model was com- parable to that generally observed in inflamed skin of AD patients (Fig. 4C).

Discussion In this report, we demonstrate that CCL18 is associated with AD. We found that CCL18 is produced by APC in the dermis

FIGURE 3. CCL18 binds to memory T cells of AD patients. A, Immu- nohistochemistry analysis of CCL18-binding cells in AD skin, faintly counterstained by hematoxylin. Original magnification, ϫ200. B, CCL18 binding on PBMC from AD patients (n ϭ 10) and healthy donors (n ϭ 5). Bars represent average Ϯ SE of CCL18 or control P300 peptide-reacting cells in the CD3ϩCLAϩ and CD3ϩCLAϩ CCR4ϩ T cell fractions. C, Phenotype analysis of human AD skin-derived T cell lines and clones. IL-4 and IFN-␥ production was analyzed by intracellular cytokine staining after stimulation with anti-CD3 and PMA. All examined T cell lines and clones bound fluorescently labeled CCL18 (red line) and CCL22 (blue line) but not control P300 peptide (black line). The percentages of CCL18bright cells are indicated in red. D, Competition binding for CCL18 on T cells. CCL18 binding (black line) could be competed by simultaneous incubation with unlabeled CCL18 (blue line), but not by CCL11 (green line) or CCL22 (orange line). Unspecific binding with control P300 peptide is shown as a gray line. E, Costaining of CCL18 and CCR3 or CCR4 on AD-derived T cells. CCR4 but not CCR3 expression in the whole cell population is dem- onstrated in the dot plots by a shift in mean fluorescence intensity (MFI). The histogram shows mean fluorescence intensity of CCL18-binding T cells (blue gate in dot plots) stained with control mIgG1 (black), CCR3 (blue), and CCR4 (red) demonstrating that the CCL18-binding cells are an homogeneous CCR4ϩ population. The Journal of Immunology 1727

healthy individuals. The mechanisms causing those differences are not known. It could be that APC from AD patients are already primed in vivo by Th2 cytokines. However, other ex- planations, including genetic factors predisposing for atopy, cannot be excluded. Our observations are novel and in line with other studies reporting notable differences between APC from atopic patients and healthy donors (23, 24). CCL18 levels in the sera of AD patients were 3-fold higher than in normal individuals. Serum levels of CCL18 increased with the transition from mild to severe atopic dermatitis disease states cor- relating with the levels of CCL22, a chemokine that has been pre- viously associated with AD (13). However, CCL18 levels were down-regulated in patients with very severe disease scores, whereas CCL22 serum levels were further increased, suggesting a differential role of each of these chemokines in AD. IFN-␥-pro- ducing Th cells are also detected in the skin lesions of severe/ chronic AD (11). Therefore, it is tempting to speculate that in these situations, when the microenvironment is less rich in IL-4 and ␥

contains IFN- , lower amounts of CCL18 are produced. This Downloaded from could account for the reduction in CCL18 expression observed in the serum of patients with the highest clinical disease scores, sug- gesting that CCL18 plays a role in the early pathogenesis of AD. A report by Hieshima et al. (3) described that CCL18 is che- moattractive for activated T cells. However, these authors did not

differentiate between naive or memory T cell subpopulations and, http://www.jimmunol.org/ therefore, the original report by Adema et al. (2) using naive T cells has led to the current belief that CCL18 is mostly a chemoat- tractant for naive T cells. In agreement with this report, we found that CCL18 binds to a distinct population of human naive CD45RAϩ, CCR7high, CD62Lhigh, CD25Ϫ, CD4ϩ, or CD8ϩ T cells in PBMC of normal individuals (data not shown). In contrast, in this study we demonstrate that CCL18 also binds to memory T cell lines and T cell clones isolated from the skin of AD patients. The binding was specific, since it could be competed with unla- by guest on October 2, 2021 beled CCL18 but not with other chemokines. CCL18 binding was also functionally relevant since CLAϩ skin-homing memory T cells reacting with CCL18 could be demonstrated in the circulation of both AD patients and normal individuals, and AD-derived mem- ory T cells migrated in vitro and in vivo in response to CCL18 alone. The recruitment of Th cells into atopic skin lesions has been attributed to the concerted action of the CCR4 ligands CCL17 and FIGURE 4. AD-derived memory T cells migrate in response to CCL18 in vitro and in vivo. A, T cells derived from AD skin responded signifi- CCL22 and the CCR10 ligand CCL27 (14, 25–28). In this study, cantly (p Ͻ 0.05) in a chemotaxis chamber assay to increasing concentra- we demonstrate that CCL18 also induces homing of memory T tions of CCL18 (F) and CCL22 (Ⅺ). Migration induced by the highest cells to human skin in vivo. The expression pattern of CCL18 is concentrations of chemokines are blocked by neutralizing mAb. Each point partially overlapping with that of CCL17 and CCL22 and comple- represents average Ϯ SE of quadruplicate values. Two of 15 experiments mentary to that of CCL27 (25, 29), which is in line with the idea are shown. B, Migration of human memory T cells to human skin grafts in that individual chemokines play redundant roles and unique func- SCID-hu Skin mice. CCL18 (eight human skin grafts from eight different tions during the homing processes of various T cell populations mice), CCL22 (eight human skin grafts from eight different mice), or PBS (15). In addition, the finding that CCL18 is expressed in a Th2 (nine human skin grafts from nine different mice) were injected into human skin grafts and skin homing of memory T cells was assessed by FACS cytokine-mediated inflammatory process such as AD, but is not analysis. CCL18 induced significant migration of the T cell line NIT and present in a Th1-mediated disease like psoriasis, implies that the T cell clone 98016T.02 (Student’s t test, p Ͻ 0.05). Two of five indepen- CCL18-responding T cells represent a specialized population. This dent experiments are shown. C, Representative immunohistochemical anal- is in contrast to CCR4 ligands, which are general T cell chemoat- ysis of PBS-, CCL11-, CCL18-, and CCL22-mediated skin homing. Infil- tractants involved in Th1- and Th2-mediated skin diseases (15). ϩ trating CD3 T cells are stained in red. Original magnification, ϫ100ϫ; This concept is supported by recent microarray studies demon- hematoxylin counterstain. strating that CCL18 mRNA is up-regulated in skin lesions of AD patients compared with samples from psoriatic and normal indi- viduals (30). and epidermis of AD patients but not in skin samples from In summary, our results provide conclusive evidence linking normal individuals or psoriatic patients. Furthermore, following CCL18 protein expression with various stages of AD and demon- stimulation with IL-4, there were higher percentages of CCL18- strating the capacity of CCL18 to recruit memory T cells into producing cells present in PBMC from AD patients than in human skin in vivo. 1728 CCL18 MEDIATES SKIN HOMING OF MEMORY T CELLS

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