Eotaxin-3/CC Ligand 26 Is a Functional Ligand for CX3CR1 Takashi Nakayama, Yoshiko Watanabe, Naoki Oiso, Tomonori Higuchi, Akiko Shigeta, Nobuyuki Mizuguchi, This information is current as Fuminori Katou, Kenji Hashimoto, Akira Kawada and of September 24, 2021. Osamu Yoshie J Immunol 2010; 185:6472-6479; Prepublished online 25 October 2010;

doi: 10.4049/jimmunol.0904126 Downloaded from http://www.jimmunol.org/content/185/11/6472

References This article cites 48 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/185/11/6472.full#ref-list-1 http://www.jimmunol.org/

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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 All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Eotaxin-3/CC Chemokine Ligand 26 Is a Functional Ligand for CX3CR1

Takashi Nakayama,* Yoshiko Watanabe,† Naoki Oiso,‡ Tomonori Higuchi,* Akiko Shigeta,* Nobuyuki Mizuguchi,x Fuminori Katou,† Kenji Hashimoto,† Akira Kawada,‡ and Osamu Yoshie*

Eotaxin-3/CCL26 is a functional ligand for CCR3 and abundantly produced by IL-4–/IL-13–stimulated vascular endothelial cells. CCL26 also functions as a natural antagonist for CCR1, CCR2, and CCR5. In this study, we report that CCL26 is yet a functional ligand for CX3CR1, the receptor for fractalkine/CX3CL1, which is expressed by CD16+ NK cells, cytotoxic effector CD8+ T cells, and CD14lowCD16high . Albeit at relatively high concentrations, CCL26 induced calcium flux and in mouse L1.2 cells expressing human CX3CR1 but not mouse CX3CR1 and competed with CX3CL1 for binding to CX3CR1. In chemo- taxis assays using human PBMCs, CCL26 attracted not only but also CD16+ NK cells, CD45RA+CD272CD8+ T cells, Downloaded from and CD14lowCD16high monocytes. Intraperitoneal injection of CCL26 into mice rapidly recruited mouse eosinophils and intra- venously transferred human CD16+ NK cells into the peritoneal cavity. IL-4–stimulated HUVECs produced CCL26 and efficiently induced adhesion of cells expressing CX3CR1. Real-time PCR showed that skin lesions of psoriasis consistently contained CX3CL1 mRNA but not CCL26 mRNA, whereas those of atopic dermatitis contained CCL26 mRNA in all samples but CX3CL1 mRNA in only about half of the samples. Nevertheless, the skin lesions from both diseases consistently contained CX3CR1 mRNA at high levels. Thus, CCL26 may be partly responsible for the recruitment of cells expressing CX3CR1 in atopic dermatitis http://www.jimmunol.org/ particularly when the expression of CX3CL1 is low. Collectively, CCL26 is another agonist for CX3CR1 and may play a dual role in allergic diseases by attracting eosinophils via CCR3 and killer lymphocytes and resident monocytes via CX3CR1. The Journal of Immunology, 2010, 185: 6472–6479.

hemokines play pivotal roles in health and disease by are known to function as an agonist for CCR3: eotaxin/CCL11, controlling migration and tissue localization of specific eotaxin-2/CCL24, eotaxin-3/CCL26, RANTES/CCL5, MCP-2/ types of cells expressing their cognate receptors (1, 2). CCL8, MCP-3/CCL7, and MCP-4/CCL17 (3, 4, 8–11). Among

C by guest on September 24, 2021 Humans have .45 , which are grouped into four them, the three eotaxins are regarded as most important because subfamilies (CXC, CC, C, and CX3C) by the structural motif of of their high specificity for CCR3. We have originally identified the N-terminal conserved cysteine residues. Chemokine receptors eotaxin-3/CCL26 through a genomic sequence analysis on the belong to the seven-transmembrane G -coupled receptor vicinity of the encoding eotaxin-2/CCL24 at the human chro- family. In humans, there are at least 18 signaling chemokine re- mosome region of 7q11.23 (9). We coined this chemokine as ceptors, which are also grouped into four subfamilies based on the eotaxin-3 because of its close genomic relationship with eotaxin- subfamily of their signaling ligands (1, 2). Chemokines and their 2/CCL24 and its specificity to CCR3 (9). Independently, Shinkai receptors, especially those involved in inflammatory responses, et al. (12) have also identified CCL26 from IL-4–stimulated tend to have highly promiscuous relationships (1, 2). This is partly HUVECs by a differential display analysis. They showed that because the chemokine system has undergone rapid gene expan- CCL26 was abundantly produced by endothelial cells stimulated sions through repeated gene duplication events during the verte- with IL-4 or IL-13 (12). Furthermore, Cuvelier and Patel (13) brate evolution (1, 2). showed that CCL26 was associated on the surface of IL-4–stim- CCR3 is a CCR selectively expressed on eosinophils, basophils, ulated endothelial cells and promoted transmigration and some Th2 cells (3–7). A large number of CC chemokines especially in shear conditions. Interestingly, CCL26 is stored in Weibel–Palade bodies, the endothelial cell-specific storage gran- ules, and is rapidly released upon secretogenic stimuli (14). Sub- *Department of Microbiology and ‡Department of Dermatology, Faculty of Medicine and xLife Science Research Institute, Kinki University, Osaka-Sayama, Osaka 589- sequent studies have further shown that IL-4 as well as IL-13 in- 8511; and †Department of Oral and Maxillofacial Surgery, Hamamatsu University duces CCL26 in a wide variety cells such as fibroblasts, bronchial School of Medicine, Hamamatsu, Shizuoka 431-3192, Japan epithelial cells, airway smooth muscle cells, intestinal epithelial Received for publication December 22, 2009. Accepted for publication September cells, and epidermal keratinocytes (15–19). Thus, CCL26 is likely 21, 2010. to play a major role in the emigration and migration of eosinophils This work was supported by Grants-in-Aid for Scientific Research from the Japan and other CCR3-expressing cells in Th2-shifted conditions. In- Society for the Promotion of Science and from the Ministry of Education, Culture, Sports, Science and Technology of Japan. deed, the serum levels of CCL26 but not those of CCL11 or Address correspondence and reprint requests to Dr. Osamu Yoshie, Department of CCL24 were shown to be well correlated with the disease severity Microbiology, Kinki University Faculty of Medicine, Osaka-Sayama, Osaka 589- of atopic dermatitis (AD) (20). Similarly, CCL26 but not CCL11 8511, Japan. E-mail address: [email protected] or CCL24 was dramatically upregulated in bronchial biopsies of Abbreviations used in this paper: AD, atopic dermatitis; B2M, b2-microglobulin; asthmatic patients 24 h after allergen challenge (21). Furthermore, PTX, pertussis toxin; SEAP, secreted form of placental alkaline phosphatase. CCL26 but not CCL11 or CCL24 was highly elevated in the blood Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 and affected tissues in Churg–Strauss syndrome, which is charac- www.jimmunol.org/cgi/doi/10.4049/jimmunol.0904126 The Journal of Immunology 6473 terized by excessive eosinophil accumulation in peripheral blood spectrophotometer (Hitachi, Tokyo, Japan) and stimulated with each and affected tissues and development of granulomatous vasculitic recombinant human chemokine. Emission fluorescence at 510 nm was organ damage (22). CCL26 has also been shown to be highly measured upon excitation at 340 and 380 nm, and the fluorescence in- tensity ratio (R340/380) was obtained. expressed in the lesions of eosinophilic esophagitis, an emerging disorder with a poorly understood pathogenesis (23, 24), and a Chemotaxis assay single-nucleotide polymorphism of the CCL26 gene is signifi- cantly associated with the disease susceptibility (23). Collectively, The chemotaxis assay was performed using Transwell plates with 5-mm pore polycarbonate membrane filters (Corning, Corning, NY) as described CCL26 appears to have a highly important role in allergic and previously (31). Cells migrated into the lower wells were lysed with 0.1% other pathological conditions where tissue infiltration of eosino- Triton X-100 (Wako, Osaka, Japan) and quantified using PicoGreen phils is prominent. Unfortunately, however, the mouse CCL26 dsDNA reagent (Molecular Probes). gene is a pseudogene (2), making it impossible to study the in vivo functions of CCL26 using mouse models. Binding assay CCL26 is a relatively low-affinity agonist for CCR3 with a po- Soluble human CX3CL1 fused with the secreted form of placental alkaline tency ∼10-fold less than that of eotaxin/CCL11 (9). Furthermore, phosphatase (CX3CL1–SEAP) was produced as described previously (32). CCL26 has been shown to act as a natural antagonist for CCR1, For binding experiments, 2 3 105 cells were incubated at 16˚C for 1 h with CCR2, and CCR5 (25, 26). These findings indicate that CCL26 is 1 nM CX3CL1–SEAP without or with increasing concentrations of com- petitors in 200 ml RPMI 1640 + 20 mM HEPES (pH 7.4) + 0.1% BSA. a less well fitting but widely interacting ligand for multiple che- Cells were washed and lysed in 50 ml 1% Triton X-100 in 10 mM Tris-HCl mokine receptors. During a thorough reexamination of various (pH 8). Cell lysates were heated at 65˚C for 10 min to inactivate cellular human chemokines on their reactivity on the panel of murine L1.2 phosphatases and centrifuged to remove cell debris. Alkaline phosphatase Downloaded from cell lines stably expressing human chemokine receptors (CCR1 to activity in 10 ml lysate was determined by chemiluminescence using the 10, CXCR1 to 7, XCR1, and CX3CR1), we unexpectedly found AURORA AP (ICN Biomedicals, Costa Mesa, CA). The binding data were analyzed with GraphPad PRISM (GraphPad Software, San Diego, that CCL26 functions as an agonist for CX3CR1, the receptor CA). for fractalkine/CX3CL1 (27), and thus efficiently attracts cells ex- pressing CX3CR1 in vitro and in vivo. CX3CR1 is known to be Flow cytometry high mainly expressed by CD16 NK cells, terminally differentiated http://www.jimmunol.org/ + Anti-CX3CR1 (clone 2A9-1) labeled with FITC or PE was purchased from cytotoxic CD8 T cells, and monocytes (27, 28). Furthermore, MBL International (Woburn, MA). Unlabeled anti-CCR3 (61828) was Geissmann et al. (29) have recently shown that the expression purchased from R&D Systems. Allophycocyanin-labeled anti-CD4 level of surface CX3CR1 subdivides blood monocytes into two (13B8.2), PerCP-Cy5.5–labeled anti-CD16 (3G8), FITC-labeled control major functional subsets; namely, CD14+CD162CX3CR1low inflam- mouse IgG2a, and allophycocyanin-labeled control mouse IgG1 were matory monocytes and CD14lowCD16+CX3CR1high homeostatic purchased from Beckman Coulter (Marseille, France). FITC-labeled anti- CD27 (LT27) was purchased from Serotec (Hokkaido, Japan). PE-labeled monocytes. Taken together, CCL26 could be a particularly ver- anti-CD45RA (HI100), PerCP-Cy5.5–labeled anti-CD19 (SJ25C1), and satile chemokine, being a dual agonist for CCR3 and CX3CR1 FITC-labeled anti-CD14 (M5E2) were purchased from BD Biosciences. and a triple antagonist for CCR1, CCR2, and CCR5. Isotype-matched control Abs were also purchased from BD Biosciences.

Cells were suspended in ice-cold PBS containing 2% FCS and 0.05% by guest on September 24, 2021 sodium azide (staining buffer) and treated with normal human serum for 20 min to block the Fc receptors. After washing, cells were incubated for 30 Materials and Methods min with the mixture of FITC-labeled mAb, PE-labeled mAb, and Cells and tissues allophycocyanin-labeled mAb. In some experiments, cells were stained first with a nonlabeled primary mAb and then with FITC-, PE-, or A mouse pre- line L1.2 was a kind gift of E. Butcher (Stanford allophycocyanin-labeled secondary Abs (Cedarlane, Ontario, Canada). University School of Medicine, Stanford, CA). The panels of L1.2 trans- After washing, cells were immediately analyzed on FACSCalibur (BD fectants stably expressing the whole set of human chemokine receptors were Biosciences) using appropriate gatings. generated by using a retroviral vector pMX-IRES-EGFP as described previously (30). L1.2 cells were infected with the recombinant viruses, and cells expressing the EGFP marker were sorted using FACSVantage SE CX3CR1 internalization (BD Biosciences, Mountain View, CA). HUVECs were purchased from Cells were suspended at 106 cells/ml in RPMI 1640 + 20 mM HEPES (pH Sankyo Junyaku (Tokyo, Japan). Human PBMCs were isolated from 7.4) + 0.1% BSA and incubated at 37˚C for 30 min with CCL26 or heparinized venous blood obtained from healthy adult donors by using the CX3CL1. After washing with PBS, the surface expression of CX3CR1 was density gradient centrifugation on Ficoll-Paque (Amersham Biosciences, determined by flow cytometry using FACSCalibur (BD Biosciences). Piscataway, NJ). Negative selection with the IMagnet System (BD Bio- sciences) was used to prepare CD8+ T cells (purity, .96%) and CD16+ CD56+ NK cells (purity, .95%) from PBMCs. Human eosinophils (purity, RT-PCR . 95%) were isolated as described previously (31). In brief, buffy coat was Total RNAs were prepared from cells and tissue sections using TRIzol prepared from heparinized venous blood by dextran T500 sedimentation reagent (Invitrogen Life Technologies) and further purified using RNeasy and subjected to a density centrifugation on 1.088 g/ml Percoll (Pharmacia Kit (Qiagen, Hilden, Germany) and RNeasy FFPE Kit (Qiagen), re- Biotech, Uppsala, Sweden). Eosinophils were further purified by negative spectively. Total RNAs (1 mg) were reverse transcribed using oligo(dT)18 selection using anti-CD16–bound micromagnetic beads and the IMagnet primer and SuperScript II reverse transcriptase (Invitrogen Life Technol- System. Skin tissues were obtained from patients with dermatological ogies). Resulting first-strand DNAs (equivalent to 20 ng total RNAs) and diseases with written informed consent. The severity of AD was assessed original total RNAs (20 ng) were amplified in a final volume of 20 ml by the criteria of Hanifin and Rajka. This study was approved by the containing 10 pmol of each primer and 1 U Ex-Taq polymerase (Takara ethical committee of Kinki University Faculty of Medicine (Osaka, Japan). Bio, Kyoto, Japan). The amplification conditions consisted of denaturation at 94˚C for 30 s (5 min for the first cycle), annealing at 60˚C for 30 s, and Calcium mobilization assay extension at 72˚C for 30 s (5 min for the last cycle) for 33 cycles for CCL26 and CX3CL1 and 27 cycles for GAPDH. The amplification Recombinant human chemokines were all purchased from R&D Systems products (10 ml each) were subjected to electrophoresis on 2% agarose and (Minneapolis, MN). Human CCL26 and CX3CL1 were also purchased stained with ethidium bromide. The primers used were +59-CTCCTGA- from PeproTech (Rocky Hill, NJ). Pertussis toxin (PTX) was purchased GTCTCCACCTTGGAA-39 and 259-TCACAATTGTTTCGGAGTTTTCA-39 from Invitrogen Life Technologies (Carlsbad, CA). Cells were suspended for CCL26; +59-GCCATGTTCACCTACCAGAGCCTCCA-39 and 259-TG- at 106 cells/ml in HBSS containing 1 mg/ml BSA and 10 mM HEPES (pH GAAGGTGGAGAATGGTCAAGGCTG-39 for CX3CL1; +59-GCCAAGG- 7.4) and loaded with 3 mM fura 2-AM fluorescence dye (Molecular Probes, TCATCCATGACAACTTTGG-39 and 259-GCCTGCTTCACCACCTTC- Eugene, OR). After washing, cells were placed on an F2000 fluorescence TTGATGTC-39 for GAPDH. 6474 CCL26 AS AN AGONIST FOR CX3CR1

ELISA 106 CFSE-labeled NK cells suspended in 200 ml PBS. After 14 h, mice were injected i.p. with 400 ml PBS or PBS containing 5 mg human CCL26 and CX3CL1 in culture supernatants were measured using ELISA CX3CL1 or CCL26. After 6 h, mice were sacrificed by cervical disloca- kits (R&D Systems). For standardization of the assays, serially diluted tion, and 6 ml PBS containing 10% FCS and 10 mM EDTA was injected recombinant CCL26 or CX3CL1 were always included in each assay. into the peritoneal cavity. After gentle massage, PBS was recovered from Cell adhesion assay each mouse. CFSE-labeled human NK cells were counted using FACS- Calibur (BD Biosciences). Smears were made on glass slides and stained Recombinant human were purchased from R&D Systems. with May–Gru¨nwald and Giemsa solutions. Eosinophil numbers were HUVECs were plated at 1 3 104 cells/well in a 96-well plate and treated obtained by counting at least 500 cells per slide. All animal experiments with IL-4 (20 ng/ml) or IFN-g (100 ng/ml) for 24 h. After replacing the were performed in accordance with the guideline of the Center of Animal medium, HUVECs in each well were incubated with 1 3 105 cells labeled Experiments, Kinki University Faculty of Medicine (Osaka, Japan). with PKH26 (Sigma-Aldrich, St. Louis, MO) for 20 min at 37˚C. After gentle washing, cells adhering to HUVECs were released by trypsin-EDTA Quantitative PCR and counted on FACSCalibur (BD Biosciences). Quantitative real-time PCR was carried out using TaqMan kit and the 7700 Cell mobilization in mice Sequence Detection System (Applied Biosystems, Foster City, CA). The conditions for PCR were 50˚C for 2 min, 95˚C for 10 min, and then 50 BALB/c mice, male and 9 wk old, were purchased from CLEA (Tokyo, cycles of 95˚C for 15 s (denaturation) and 60˚C for 1 min (annealing Japan) and kept in specific pathogen-free conditions for at least 1 wk before extension). The primers and fluorogenic probes for CCL26, CX3CL1, + . experiments. Human CD16 NK cells (purity, 95%) were labeled with 2 CX3CR1, and b2-microglobulin were obtained from TaqMan kits (Applied mM CFSE (Molecular Probes) for 15 min. Mice were injected i.v. with 1 3 Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021

FIGURE 2. CCL26 induces chemotaxis via CX3CR1. A, Dose-response FIGURE 1. CCL26 induces calcium mobilization via CX3CR1. A, experiments. L1.2 cells transfected with the vector only (L1.2-vector), CCL26 induces calcium mobilization in L1.2 cells expressing CCR3 stably expressing CCR3 (L1.2-CCR3) or stably expressing CX3CR1 (L1.2-CCR3) and those expressing CX3CR1 (L1.2-CX3CR1). Cells were (L1.2-CX3CR1), were examined for cell migration to indicated concen- loaded with fura 2-AM and stimulated with CCL11, CX3CL1, and CCL26 trations of CCL26 and CX3CL1. B, A checkerboard-type analysis. L1.2- at indicated concentrations (5 3 105 cells per assay). Intracellular calcium CX3CR1 cells were examined for cell migration with CCL26 added to mobilization was measured on a fluorescence spectrophotometer. Pre- upper and/or lower wells at 100 nM as indicated. C, Inhibition by PTX. treatment of L1.2-CX3CR1 with PTX at 500 ng/ml was performed at 37˚C L1.2-CX3CR1 cells were pretreated at 37˚C for 30 min without or with for 30 min. Representative results from three separate experiments are PTX at 500 ng/ml and examined for cell migration to CCL26 at 100 nM. shown. B, Desensitization experiments. L1.2-CX3CR1 cells were loaded D, Additive effects of CCL26 and CX3CL1. L1.2-CX3CR1 cells were with fura 2-AM and sequentially stimulated with CCL26 and CX3CL1 as examined for cell migration to indicated concentrations of CCL26 without indicated. Intracellular calcium mobilization was measured on a fluores- (open columns) or with 1 nM of CX3CL1 (closed columns). Cell migration cence spectrophotometer. Representative results from three separate experi- was expressed as percentage input cells. Results from three separate ments are shown. experiments are shown as mean 6 SEM. *p , 0.05. The Journal of Immunology 6475

Biosystems). Quantification of the was performed using the Sequence Detector System Software (Applied Biosystems). Statistical analysis Statistical significance was determined using Student t test. We considered p , 0.05 as statistically significant.

Results CCL26 induces calcium mobilization via CX3CR1 To reevaluate the receptor usage of various human chemokines, we tested their ability to induce calcium mobilization using a panel of mouse L1.2 cell lines stably expressing all the known human che- mokine receptors (CCR1–10, CXCR1–7, XCR1, and CX3CR1). By testing a total of 32 recombinant human chemokines (CCL1, -2, -3, -4, -5, -7, -8, -11, -13, -14, -15, -16, -17, -18, -19, -20, -21, -22, -24, -25, -26, -27, -28; CXCL4, -8, -9, -10, -12, -13, -16; XCL1; CX3CL1), we found that CCL26 was able to induce cal- cium mobilization via not only CCR3 but also CX3CR1 in a

PTX-sensitive manner (Fig. 1A). This reactivity was specific for Downloaded from CCL26, as other eotaxins (eotaxin/CCL11 and eotaxin-2/CCL24) did not induce such responses in L1.2-CX3CR1 (data not shown). FIGURE 4. CX3CR1 internalization. L1.2-CX3CR1 cells were in- In desensitization experiments, ∼10-fold excess concentrations of cubated with indicated concentrations of CCL26 or CX3CL1 at 37˚C for CCL26 were required to desensitize CX3CR1 to CX3CL1 effec- 30 min. The surface expression of CX3CR1 was determined on FACS- tively, whereas CX3CL1 at equal concentrations completely de- Calibur. Representative results from two separate experiments are shown. ∼ sensitized CX3CR1 to CCL26 (Fig. 1B). Thus, CCL26 was 10- http://www.jimmunol.org/ fold less in potency than CX3CL1. It was also noted that the motaxis, not chemokinesis, in L1.2-CX3CR1 cells (Fig. 2B). Che- calcium mobilization induced by CCL26 was transient compared motaxis induced in L1.2-CX3CR1 cells by CCL26 was also sensi- with that induced by CX3CL1. tive to PTX, confirming the Gai-coupled signaling (Fig. 2C). Fur- CCL26 induces chemotaxis via CX3CR1 The above results prompted us to examine the ability of CCL26 to induce chemotaxis via CX3CR1. As shown in Fig. 2A, CCL26 induced cell migration in both L1.2-CCR3 and L1.2-CX3CR1

with peak responses at ∼100 nM, and CX3CL1 induced cell mi- by guest on September 24, 2021 gration in L1.2-CX3CR1 with a peak response at 10 nM. Notably, although the potency of CCL26 was ∼10-fold less than that of CX3CL1, its efficacy was comparable with that of CX3CL1. The checkerboard-type analysis confirmed that CCL26 induced che-

FIGURE 5. CCL26 induces cell migration in PBMCs via CCR3 and CX3CR1. Human PBMCs were examined for cell migration to CCL26 (d) and CX3CL1 (s) at indicated concentrations. CCL19 (♦) at 100 ng/ml and FIGURE 3. Specific binding of CCL26 to CX3CR1. L1.2 cells stably CCL2 (n) at 10 ng/ml were used as positive controls. After incubation for 2 expressing CX3CR1 (L1.2-CX3CR1) (A) or human CD16+ NK cells (B) h at 37˚C, input cells and cells migrated into lower wells were stained for were incubated with 1 nM CX3CL1–SEAP and increasing concentrations various surface markers as indicated and quantified by flow cytometry. of CX3CL1 (s) or CCL26 (d) at 16˚C for 1 h. After washing, the amount Eosinophils were purified from PBMCs and used for chemotaxis assays. of cell-bound CX3CL1–SEAP was determined enzymatically. Results Cell migration was expressed as percentage input cells. Results from three from three separate experiments are shown as mean 6 SEM. separate experiments are shown as mean 6 SEM. 6476 CCL26 AS AN AGONIST FOR CX3CR1

thermore,CCL26andCX3CL1wereadditiveininductionofcell migration in L1.2-CX3CR1 cells (Fig. 2D). Specific binding of CCL26 to CX3CR1 Previously, we used soluble CX3CL1 tagged with the secreted form of alkaline phosphatase (CX3CL1–SEAP) to quantify its specific binding to CX3CR1 (32). Therefore, we examined specific bind- ing of CCL26 to CX3CR1 through its competition with CX3CL1– SEAP. As shown in Fig. 3A, the specific binding of 1 nM CX3CL1–SEAP to L1.2-CX3CR1 was dose-dependently inhibited by unlabeled CX3CL1 and CCL26 with an IC50 of 2.4 nM and 35 nM, respectively. Furthermore, the specific binding of 1 nM CX3CL1–SEAP to human CD16+ NK cells was also competed by unlabeled CX3CL1 and CCL26 with an IC50 of 1.0 nM and 17 nM, respectively (Fig. 3B). Thus, the binding affinity of CCL26 to CX3CR1 was 10- to 20-fold less than that of CX3CL1, this in keeping with its ~10-fold lower potency in induction of calcium flux and chemotaxis in L1.2-CX3CR1 cells than that of CX3CL1

(Figs. 1 and 2). Downloaded from CX3CR1 internalization In general, the binding of a chemokine to its receptor rapidly induces receptor internalization, which is important in the regulation of receptor signaling activity. Therefore, we next examined CX3CR1 internalization by CCL26 and CX3CL1. The surface expression http://www.jimmunol.org/ of CX3CR1 was determined before and after treatment with CCL26 or CX3CL1. As shown in Fig. 4, CX3CL1 induced a con- centration-dependent internalization of CX3CR1, whereas no in- ternalization was observed in the cells treated with CCL26, even at concentrations that induced the maximum chemotactic respon- ses. This observation may also be in keeping with the low affinity of CCL26 to CX3CR1. Any qualitative differences in the signaling pathway activated by CX3CL1 and CCL26 remain to be seen.

Chemotactic response of cells expressing CX3CR1 in human by guest on September 24, 2021 PBMCs to CCL26 In PBMCs, CCR3 is mainly expressed by eosinophils (3, 4), whereas CX3CR1 is highly expressed by CD16+ NK cells, CD45RA+CD27– CD8+ terminally differentiated cytotoxic T cells, and CD14low CD16high homeostatic monocytes (27–29). We therefore compared chemotactic responses of human PBMCs to CCL26 and CX3CL1 (Fig. 5). As expected, CX3CL1 selectively induced vigorous mi- gration in CD45RA+CD272CD8+ T cells, CD16+ NK cells, and CD14lowCD16high monocytes. Furthermore, although at relatively high concentrations, CCL26 induced vigorous migration not only in eosinophils but also in CD45RA+CD27–CD8+ T cells, CD16+ NK cells, and CD14lowCD16high monocytes with an efficacy quite FIGURE 6. CCL26 is abundantly produced by IL-4–stimulated similar to that of CX3CL1. HUVECs and induces cell adhesion via CX3CR1. A,RT-PCRanalysis. HUVECs were stimulated without or with IL-1b (10 ng/ml), TNF-a IL-4–stimulated HUVECs abundantly secrete CCL26 and (50 ng/ml), IFN-g (100 ng/ml), IL-4 (20 ng/ml), and their combinations for efficiently induce adhesion of CX3CR1-expressing cells 24 h, and total RNAs were prepared. PHA-stimulated PBMCs were used as As reported previously, HUVECs expressed and secreted CCL26 positive control. Reverse transcription-PCR analysis was performed for upon IL-4 treatment (12, 13), whereas HUVECs expressed and CCL26, CX3CL1, and GAPDH. Representative results from two separate secreted CX3CL1 upon treatment with proinflammatory cytokines experiments are shown. B, ELISA assay. HUVECs were seeded in tripli- such as IL-1b, TNF-a, and IFN-g (33, 34) (Fig. 6A,6B). Fur- cate in 24-well plates and cultured without or with indicated cytokines for 24 h. The contents of CCL26 and CX3CL1 secreted in the culture thermore, IFN-g suppressed the induction of CCL26 by IL-4, and supernatants were measured by ELISA. Data are shown as mean 6 SEM IL-4 suppressed the induction of CX3CL1 by IL-1b, TNF-a, and from three separate experiments. C and D, Cell adhesion assay. HUVECs IFN-g. Thus, CX3CL1 and CCL26 are produced by HUVECs in were stimulated without or with IFN-g (100 ng/ml) or IL-4 (20 ng/ml) for 24 h. Test cells were labeled with PKH26. Adhesion assays were per- formed as described in Materials and Methods. For blocking experiments, and those stably expressing CX3CR1 (L1.2-CX3CR1; closed columns) HUVECs were pretreated with 10 mg/ml anti-CX3CL1 or anti-CCL26 for were used for the adhesion assay. D, CD16+ NK cells prepared from hu- 20 min. L1.2-vector and L1.2-CX3CR1 were pretreated with 10 mg/ml man PBMCs (purity, .95%) were used for the adhesion assay. Cell ad- anti–LFA-1 (anti-integrin aL) or anti–VLA-4 (anti-integrin a4) for 20 min. hesion was expressed as percentage input cells. Results from three separate C, L1.2 cells transfected with the vector only (L1.2-vector; open columns) experiments are shown as mean 6 SEM. *p , 0.01. The Journal of Immunology 6477 a highly reciprocal manner in accordance with the Th1 and Th2 CCL26 is a pseudogene (2, 36), we first examined whether hu- conditions, respectively. It was also notable that the amounts of man CCL26 was capable of inducing signals via mouse CCR3 CCL26 secreted by IL-4–treated HUVECs (∼40 ng/ml) were (mCCR3) and mCX3CR1. We found that CCL26 was chemotactic .10-fold those of CX3CL1 secreted by IFN-g–treated HUVECs for L1.2 cells expressing mCCR3 but not for those expressing (∼1.5 ng/ml). Thus, the lower potency of CCL26 on CX3CR1 may mCX3CR1 (data not shown). Therefore, we first injected CFSE- be easily compensated by its higher production than that of labeled human CD16+ NK cells into mice via the tail vein. After CX3CL1. 14 h, we injected chemokines into the peritoneal cavities of the Chemokines are known to induce firm adhesion of cells ex- mice. As shown in Fig. 7, human CCL26 recruited both human pressing their cognate receptors to vascular endothelial cells via NK cells and endogenous mouse eosinophils into the peritoneal transient activation of LFA-1 or VLA-4 (1, 35). However, the cavity, and human CX3CL1 recruited human NK cells. Anti-CCL26 membrane-anchored form of CX3CL1 is known to induce firm suppressed the recruitment of both human NK cells and mouse adhesion of CX3CR1-expressing cells even without activation of integrins (27). Therefore, we next examined adhesion of CX3CR1-expressing cells to HUVECs treated with IFN-g or IL-4. As shown in Fig. 6C, we observed enhanced adhesion of L1.2- CX3CR1 not only to IFN-g–treated HUVECs but also to those treated with IL-4. The enhanced adhesion of L1.2-CX3CR1 to IFN-g–treated HUVECs and those treated with IL-4 was sig- nificantly inhibited by anti-CX3CL1 and anti-CCL26, respec- Downloaded from tively. However, anti–LFA-1 or anti–VLA-4 selectively inhibited the adhesion of L1.2-CX3CR1 to IL-4–treated HUVECs but not to IFN-g–treated HUVECs. Thus, CCL26-mediated adhesion of L1.2-CX3CR1 to HUVECs was dependent on the activation of these integrins. We also confirmed enhanced adhesion of human + CD16 NK cells to both IFN-g–treated and IL-4–treated HUVECs http://www.jimmunol.org/ (Fig. 6D). Again, their adhesion to IFN-g–treated HUVECs and IL-4–treated HUVECs was effectively suppressed by anti- CX3CL1 and anti-CCL26, respectively. In vivo recruitment of CCR3- and CX3CR1-expressing cells by CCL26 We next wished to demonstrate attraction of CX3CR1-express- ing cells by CCL26 in vivo. Because the mouse homologue of by guest on September 24, 2021

FIGURE 8. Coexpression of CCL26 and CX3CR1 in AD. A, Quanti- tative real-time PCR analysis for the expression of CCL26, CCR3, CX3CL1, and CX3CR1 mRNAs in various skin tissues. Total RNAs were prepared from formalin-fixed, paraffin-embedded sections from normal skin tissues (n = 5), skin tissues from psoriasis patients (n = 5), and skin FIGURE 7. In vivo recruitment of cells expressing CCR3 and CX3CR1 tissues from AD patients (n = 13). Real-time PCR was performed to by CCL26. Male BALB/c mice were i.v. injected with CFSE-labeled hu- quantify mRNA expression of CCL26, CCR3, CX3CL1, and CX3CR1. + man CD16 NK cells (purity, .95%). After 14 h, mice were i.p. injected The results are normalized by the expression of the b2-microglobulin gene with recombinant human CX3CL1 or CCL26. After 6 h, mice were sac- (B2M). Representative results from two separate experiments are shown. rificed, and peritoneal lavage fluids were collected. CFSF-labeled human B, Correlation analysis between the level of mRNA expression of CCL26, NK cells were counted by flow cytometry, and mouse eosinophils were CCR3, CX3CL1, and CX3CR1 and the severity of AD. The AD patients counted by staining with May–Gru¨nwald and Giemsa solutions on the were grouped into two groups according to the criteria of Hanifin and slides. Results from three separate experiments are shown as mean 6 SEM. Rajka. *p , 0.05. 6478 CCL26 AS AN AGONIST FOR CX3CR1 eosinophils by CCL26, and anti-CX3CL1 suppressed the re- affinity and potency of CCL26 for CX3CR1 are ~10-fold less than cruitment of human NK cells by CX3CL1. those of CX3CL1, the sheer abundance of CCL26 produced by IL- 4– or IL-13–stimulated cells may easily compensate for its low Dual role of CCL26 in AD affinity and potency as the CX3CR1 agonist. CCR3 is considered AD is a Th2-dominant disease, whereas psoriasis a Th1-mediated to be involved in Th2-mediated diseases including AD and asthma disease (37, 38). It has been shown that CX3CL1 mRNA is highly (41, 42), whereas CX3CR1 is reported to be involved in Th1- elevated in the skin lesion of psoriasis (34). By using quantitative mediated diseases such as rheumatoid arthritis, diabetes, lichen real-time PCR, we compared mRNA levels of CCL26, CCR3, planus, and psoriasis (34, 43, 44). Furthermore, CCL26 and CX3CL1, and CX3CR1 in normal, psoriasis, and AD skin tissues. CX3CL1 are reciprocally induced in vascular endothelial cells by As shown in Fig. 8A, CCL26 mRNA and CCR3 mRNA were Th1 and Th2 cytokines, respectively (12, 33, 34). Thus, CCL26 consistently detected at high levels in AD skin samples. In con- and CX3CL1 are likely to have a highly reciprocal role in blood trast, CX3CL1 and CX3CR1 transcripts were consistently detected cell–endothelial interactions in Th2 and Th1 conditions, respec- at high levels in psoriasis skin samples. Notably, CX3CR1 mRNA tively. However, from the current results, CCL26 may also have was also consistently detected at high levels in AD skin tissues, a role in extravasation of CX3CR1-expressing cells in Th2-shifted although mRNA of its ligand CX3CL1 was detected in only half of conditions. We have indeed demonstrated that although CX3CL1 the samples. Thus, in AD cases without high CX3CL1 expression, mRNA was not always detected, the mRNA levels of CX3CR1 CCL26 might be responsible for the attraction of cells expressing were consistently augmented together with those of CCL26 in CX3CR1. Unfortunately, we were unable to localize the cells AD skin lesions (Fig. 8). Thus, the extravasation of CX3CR1- expressing CCL26 or CX3CR1 in situ in AD skin tissues due to the expressing cells into AD skin tissue may be mediated by CCL26 Downloaded from lack of usable Abs. in Th2-shifted conditions. We also analyzed the correlation of AD disease severity with the Recently, it has been suggested that cytotoxic effector lym- mRNA levels of CCL26, CCR3, CX3CL1, and CX3CR1. The phocytes are also involved in the pathogenesis of allergic diseases disease severity was significantly correlated with the levels of such as AD and asthma (45–47). Yawalkar et al. (45) reported that CX3CL1 mRNA but not those of CCL26 mRNA (Fig. 8B). Thus, cytotoxic effector lymphocytes existed in AD and psoriasis skin at

IL-4–induced CCL26 may play a minor role in AD disease se- the same levels, suggesting that cell-mediated cytotoxic mecha- http://www.jimmunol.org/ verity, even though it attracts both eosinophils and the cells also nisms could be involved in epidermal cell injury of AD. Fur- attracted by CX3CL1. This may indicate that the shift to Th1 may thermore, Echigo et al. (48) reported that infiltration of CX3CR1- be responsible for the disease severity of AD (37, 39). However, it expressing lymphocytes was increased in skin tissues of not only should also be reminded that CX3CL1 is inducible not only by the psoriasis but also AD, CX3CL1 was expressed in endothelial cells Th1-type IFN-g but also by the proinflammatory cyto- in chronic skin lesions of AD, and serum CX3CL1 levels corre- kines such as IL-1b and TNF-a (33, 34), and the disease severity lated with the disease severity. In the current study, we have also of AD was reported to correlate with the serum levels of TNF-a shown that, although CX3CR1 mRNA expression is consistently (40). Thus, the increased levels of CX3CL1 in AD skin tissues increased in AD skin, CX3CL1 mRNA levels are increased in may be indirectly correlated with disease severity through the ex- only a fraction of cases with a significant correlation with the by guest on September 24, 2021 pression levels of TNF-a. disease severity (Fig. 8). Thus, CCL26 may be responsible for the infiltration of cells expressing CX3CR1 in AD skin where Discussion CX3CL1 expression is low. CCL26-induced infiltration of cells Eotaxin-3/CCL26 was identified as the third member of the eotaxin expressing CX3CR1 may promote the shift from Th2 to Th1, re- family (9, 12), a group of CC chemokines that are regarded to be sulting in production of CX3CL1. highly specific for CCR3. Because CCR3 is selectively expressed Collectively, CCL26 is another agonist for CX3CR1. Thus, it on eosinophils, basophils, and some Th2 cells (3–7), the eotaxins may play a dual role in the pathogenesis of allergic and other dis- are considered to play the major roles in allergic diseases. Among eases such as eosinophilic esophagitis and Churg–Strauss syndrome the eotaxins, however, CCL26 appears to be particularly important by attracting not only CCR3-expressing cells but also CX3CR1- in allergic diseases (20, 21). Furthermore, Ogilvie et al. (25) have expressing cells (22–24). The role of cells expressing CX3CR1 demonstrated that CCL26 is a natural antagonist for CCR2 and such as terminally differentiated CD8+ killer T cells and CD16+ promotes active movement of monocytes away from a CCL26 NK cells in the pathogenesis of allergic diseases, eosinophilic gradient especially in synergy with an additional gradient of esophagitis, and Churg–Strauss syndrome remains to be seen. CCL2, although the mechanism by which CCL26 exerts this re- pellent effect in a PTX-sensitive manner has not been defined. Acknowledgments Moreover, Petkovic et al. (26) have reported that CCL26 acts as We thank Kunio Hieshima and Namie Sakiyama for helpful discussions and a natural antagonist for CCR1 and CCR5 as well. Thus, CCL26 excellent technical assistance, respectively. may have multiple roles in allergic inflammation by attracting eosinophils, basophils, and some Th2 cells via CCR3 and con- comitantly blocking the recruitment of monocytes and Th1 cells Disclosures via CCR1, CCR2, and CCR5. Furthermore, recent studies have The authors have no financial conflicts of interest. shown that CCL26 is closely associated in the pathogenesis of diseases such as eosinophilic esophagitis and Churg–Strauss syn- References drome (22–24). Thus, CCL26 may be an important diagnostic and 1. Yoshie, O., T. Imai, and H. Nomiyama. 2001. Chemokines in immunity. Adv. therapeutic target in the future. Immunol. 78: 57–110. In this study, we have demonstrated that CCL26 is yet another 2. Zlotnik, A., O. Yoshie, and H. Nomiyama. 2006. The chemokine and superfamilies and their molecular evolution. Genome Biol. 7: 243. functional ligand for CX3CR1, the fractalkine/CX3CL1 receptor, 3. Daugherty, B. L., S. J. Siciliano, J. A. DeMartino, L. Malkowitz, A. Sirotina, and which is expressed on CD16high NK cells, CD45RA+CD27–CD8+ M. S. Springer. 1996. Cloning, expression, and characterization of the human + eosinophil eotaxin receptor. J. Exp. Med. 183: 2349–2354. T cells (terminally differentiated effector CD8 T cells), and 4. Ponath, P. D., S. Qin, T. W. Post, J. Wang, L. Wu, N. P. Gerard, W. Newman, low high CD14 CD16 homeostatic monocytes (27–29). Although the C. Gerard, and C. R. Mackay. 1996. Molecular cloning and characterization of The Journal of Immunology 6479

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