International Immunology, Vol. 27, No. 4, pp. 169–181 © The Japanese Society for Immunology. 2014. All rights reserved. doi:10.1093/intimm/dxu098 For permissions, please e-mail: [email protected] Advance Access publication 25 October 2014

CCR8 regulates contact hypersensitivity by restricting cutaneous dendritic cell migration to the draining lymph nodes

Rikio Yabe1,2,3,*, Kenji Shimizu1,2,*, Soichiro Shimizu2, Satoe Azechi2, Byung-Il Choi2, Katsuko Sudo2, Sachiko Kubo1,2, Susumu Nakae2, Harumichi Ishigame2, Shigeru Kakuta4 and Yoichiro Iwakura1,2,3,5 1Center for Animal Disease Models, Research Institute for Biomedical Sciences (RIBS), Tokyo University of Science, Noda, Chiba 278-0022, Japan 2Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, University of Tokyo (IMSUT), Minato-ku, Tokyo 108-8639, Japan 3Medical Mycology Research Center, Chiba University, Inohana Chuo-ku, Chiba 260-8673, Japan

4 A rticle

Department of Biomedical Science, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Featured Tokyo 113-8657, Japan 5Core Research for Evolutional and Technology (CREST), Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan

Correspondence to: Y. Iwakura; E-mail: [email protected] *These authors equally contributed to this work. Received 2 September 2014, accepted 17 October 2014

Abstract Allergic contact dermatitis (ACD) is a typical occupational disease in industrialized countries. Although various and are suggested to be involved in the pathogenesis of ACD, the roles of these molecules remain to be elucidated. CC 8 (CCR8) is one such molecule, of which expression is up-regulated in inflammatory sites of ACD patients. In this study, we found that Ccr8−/− mice developed severer contact hypersensitivity (CHS) responses to 2,4-dinitrofluorobenzene, a murine model of ACD, compared with wild-type mice. T cells from Ccr8−/− mice showed enhanced proliferative recall responses and Th1 and Th17 cell populations were expanded in these mice. However, CHS responses were similar between SCID mice adoptively transferred with Ccr8−/− and wild-type T cells, suggesting that CCR8 in T cells is not responsible for the exacerbation of CHS. Notably, skin- resident dendritic cells (DCs), such as Langerhans cells and dermal DCs, and inflammatory DCs were highly accumulated in lymph nodes (LNs) of Ccr8−/− mice after sensitization. Consistent with this, Ccr8−/− antigen-presenting cells readily migrated from the skin to the draining LNs after sensitization. These observations suggest that CCR8 negatively regulates migration of cutaneous DCs from the skin to the draining LNs in CHS by keeping these cells in the skin.

Keywords: CCR8, contact hypersensitivity, dermal dendritic cells, Langerhans cells, migration

Introduction The prevalence of contact allergy is ~20% in the populations resulting in the generation of effector/memory T cells in the of North America and Western Europe, and sometimes these dLNs. (ii) Elicitation phase: a second challenge of the same people develop allergic contact dermatitis (ACD), resulting haptens to sensitized individuals causes the activation of in an impairment in the quality of life (1, 2). Contact hyper- memory T cells, resulting in the elicitation of local inflamma- sensitivity (CHS), a mouse model of ACD, is triggered by tion. Thus, the migration of cutaneous DCs to dLNs during repetitive contact-exposure of low-molecular weight chemi- the sensitization phase is crucial in the development of CHS. cals termed as haptens to the skin. CHS is constituted of Langerhans cells (LCs) are a subset of DCs, which are spe- two phases (3–5). (i) Sensitization phase: exposure of skin to cifically localized in skin epidermis. Upon antigenic challenge haptens activates cutaneous dendritic cells (DCs), resulting to skin, LCs acquire antigens(s) and migrate to dLNs, where in the migration of DCs into draining lymph nodes (dLNs) in they present the antigens to naive T cells (7, 8). Dermal den- a CCR7-CCL19/CCL21-dependent manner (6). Then, these dritic cells (dDCs) are another subset of epidermis/dermis- haptenized -bearing DCs sensitize naive T cells, localized DCs (9). These DC subsets are the most important 170 CCR8 suppresses contact dermatitis antigen-presenting cells (APCs) for the immune responses in sex-matched mice were used for all experiments. All experi- the skin (9, 10). In addition, inflammatory DCs (iDCs), which ments were approved by the institutional animal use commit- are likely to be differentiated from peripheral blood-circulat- tees and were conducted according to the institutional ethical ing (11), are also suggested to be involved in guidelines for animal experiments and safety guidelines for this response (12). However, the pathological roles of each manipulation experiments. DC subset in the development of CHS are still controversial (13–18). Contact hypersensitivity In the elicitation stage, various types of pro-inflammatory CHS was induced as described previously (33). Briefly, mice cytokines and chemokines are released from immune cells were sensitized by painting the shaved abdomen with 50 µl of and keratinocytes [KCs (19, 20)]. Elevated levels of cytokines 0.5% DNFB (Tokyo chemical industry, Tokyo, Japan) in ace- and chemokines, such as IFN-γ, IL-4, TNF, IL-10, CCL1 (also tone/olive oil (4:1). Five days after the first immunization, the known as I-309 and TCA-3), are detected in the skin biopsies mice were challenged with 25 µl of 0.2% DNFB on both sur- from ACD patients sensitized with nickel (21). Notably, CC faces of the right ear. Solvent alone was applied to the left ear 8 (CCR8), the chemokine receptor for as a control. Twenty-four hours after the second challenge, a CCL1 and CCL8, was also up-regulated in the biopsies, sug- small piece of the earlobe was excised using a 6-mm punch gesting possible involvement of the CCR8–CCL1/CCL8 axis instrument and weighed. Earlobe swelling was assessed as in the pathogenesis of ACD. follows: [Increment of earlobe swelling (mg)] = [weight of The chemokine–chemokine receptor system plays impor- challenged earlobe (mg)] – [weight of vehicle-treated earlobe tant roles in the trafficking of leukocytes from the circulation (mg)]. Ear thickness was daily measured with a micrometer to specific organs or inflammatory sites (22). CCR8 induces caliper. Ear swelling is shown as a percentage of thickness chemotactic migration of cells by activating calcium influx in compared with ears at day 0. response to CCL1 (23, 24). CCR8 is expressed in monocytes and , and is involved in the development of peritoneal adhesions (25), type I diabetes (26) and hepatitis Immunohistochemistry (27) through recruitment of monocytes/macrophages to the Ear biopsies were fixed with 10% neutral formalin, dehy- drated and paraffin-embedded. About 5 µm sections were inflammatory sites. CCR8 is also expressed in hT 2 cells, and mediates and calcium mobilization of these cells stained with hematoxylin and eosin (HE).

(28). Consistent with this, in asthma, a Th2-type disorder (29), the CCR8+ population is expanded, and Ccr8−/− mice titers are refractory to experimental allergic airway inflammation Sera were collected 3 days after the second challenge and (30), suggesting that CCR8 is involved in the pathogenesis levels of IgG specific for dinitrophenyl (DNP)-BSA were of T 2-mediated diseases. Recently, Islam et al. (31) reported h measured by an ELISA-based assay. An aliquot of serum that CCL8, a newly identified CCR8 agonist, promotes atopic (50 µl) was applied to a DNP-BSA-coated 96-well plate, and dermatitis in mice by recruiting CCR8-expressing T 2 cells. h the plate was incubated at room temperature for 2 h. Then, On the other hand, CCR8 suppresses septic peritonitis by wells were washed and incubated with alkaline phosphatase- suppressing activation of macrophages (32). A regulatory conjugated goat anti-mouse IgG (Zymed, San Francisco, CA, role of CCR8 is also suggested in ACD, because CCR8- USA) at room temperature for 1 h, followed by the incubation expressing, nickel-specific skin-homing T cells produce IL-10 with substrate phosphatase Sigma 104 (Sigma–Aldrich, St. (21). Therefore, the role of CCR8 in the pathogenesis of ACD Louis, MO, USA). The absorbance at 415 nm was measured remains still controversial. using a microplate reader (MTP-300, HITACHI, Japan). In this report, we have generated Ccr8−/− mice, and found that Ccr8−/− mice were more susceptible to 2,4-dinitrofluor- obenzene (DNFB)-induced CHS compared with wild-type Recall proliferation assay (WT) mice. This was because migration of DCs from the skin Single cell suspensions were prepared from dLNs 24 h after to dLNs was enhanced in Ccr8−/− mice. These results suggest sensitization with 0.5% DNFB, and were cultured in the pres- that CCR8 regulates migration of DCs from skin to dLNs in ence of indicated concentrations of DNBS (Tokyo chemical contact allergic inflammation. industry) in 96-well plates (2 × 105 cells). PMA and ionomycin (50 and 500 ng/ml) treated cells were used as the controls. Methods After incubation for 66 h, cells were labeled with [3H]-thymidine (0.25 µCi/ml, PerkinElmer, Waltham, MA, USA) for 6 h, and Mice harvested using a Micro 96 cell harvester (Skatron, Sterling, Ccr8−/− mice were generated by homologous recombination VA, USA). [3H]-radioactivity in the acid insoluble fraction was techniques using E14.1 ES cells as described in Supporting measured using a Micro Beta Systems (Pharmacia Biotech, Materials and Methods, available at International Immunology Piscataway, NJ, USA). IFN-γ and IL-17 concentrations in Online, and backcrossed to the BALB/cA mice for 8 to 10 gen- culture supernatants after 72 h stimulation with DNBS were erations (Supplementary Figure 1 is available at International determined using OptiEIA IFN-γ (BD Pharmingen, San Diego, Immunology Online). WT and SCID mice were purchased CA, USA) and Duo set IL-17 (R&D systems, Minneapolis, MN, from CLEA Japan. Mice were bred under specific pathogen- USA), respectively, according to the manufacturer’s proto- free conditions in an environmentally controlled clean room cols. The proportions of Th and Treg cells were determined at the IMSUT, and the RIBS. Age- (8- to 12-week-old) and by flow cytometry as described below. CCR8 suppresses contact dermatitis 171 Isolation of T and B cells by magnetic cell sorter quantitative analysis, SYBR Green qPCR (TaKaRa) was T and B cells were isolated from the spleen and LNs using used with specific primer sets (Supplementary Table 3 is autoMACS (Miltenyi Biotech, Bergish Gladbach, Germany) available at International Immunology Online). with anti-CD90.2- and anti-B220-conjugated microbeads (Miltenyi Biotech), respectively, in accordance with the manu- Staining of epidermal sheets facturer’s instructions. Epidermal sheets were obtained from earlobes accord- ing to a previous report (35). Earlobes were cut off at the T cell proliferation base, and split into dorsal and ventral halves. The dorsal T cells (2 × 105 cells) were cultured in anti-CD3 (clone 145- sheets were incubated with 20 mM EDTA/PBS for 2 h at 2C11; eBioscience, San Diego, CA, USA)-coated-96-well 37°C. The epidermis was fixed in cold acetone for 20 min, plates. T-cell proliferation was determined as described permeabilized in 0.1% saponin/PBS, and blocked with above. 5% FBS/PBS. After washing, the sheets were stained with 5 µg/ml biotin-conjugated anti-CD207 monoclonal anti- body (clone 929F3.01, Dendritics, Dardilly, France), fol- Adoptive T cell transfer lowed by DyLight549-conjugated streptavidin (Jackson T cells were purified from both spleens and LNs using ImmunoResearch, West Grove, PA, USA). Sheets were 5 autoMACS with anti-CD90.2-microbeads (89% pure; 1 × 10 mounted with Mount-Quick (Daido Sangyo, Saitama, 6 or 1 × 10 ), and they were intravenously injected into recipi- Japan). Images were obtained using fluorescence micros- ent SCID mice. After 1 day, these mice were subjected to copy, BZ-9000 (Keyence, Osaka, Japan). Cell numbers DNFB-induced CHS. were counted in three independent fields per section of a In the case of DNFB-sensitized T cell transfer into WT mice, mouse epidermal sheet. mice were sensitized by painting with 0.5% DNFB. Five days after sensitization, T cells were isolated from dLNs by MACS FITC migration with anti-CD90.2-microbeads. The resulting cells (2 × 107 cells/mouse; 98% pure) were intravenously injected into WT Mice were sensitized by painting with 50 µl of 0.5% FITC mice. Six hours after injection, mice were challenged with isomer I (Sigma–Aldrich) in acetone/dibutylphthalate (1:1, DNFB. vol:vol) on the shaved back skin. After 24 h, dLNs were col- lected and a single cell suspension was prepared by digest- ing with collagenase. Cells were blocked, and were stained Flow cytometry with . The contents of FITC+ DC subsets were ana- Antibodies used for flow cytometric analysis are listed in lysed by flow cytometry. Supplementary Table 1, available at International Immunology Online. Flow cytometry was performed as described previ- Immunofluorescence staining ously (34). Briefly, cells were stained with primary antibod- LNs were embedded in OCT compound (Sakura Finetek ies for 30 min at 4°C after blocking with 2.4G2, followed by Japan, Tokyo, Japan) and frozen with liquid nitrogen. About secondary antibodies. Cells were analysed by flow cytom- 7 µm serial cryosections were fixed with 4% PFA, blocked eters, FACSCalibur (Becton Dickinson, Sparks, MD, USA) or with 5% goat serum in PBS/0.3% Triton X-100 and stained FACSCantoII (Becton Dickinson) with FlowJo (Tree Star). Dead with hamster anti-mouse CD11c antibody (clone, N418; cells were stained with either 7AAD (Sigma–Aldrich) or LIVE/ Biolegend, San Diego, CA, USA), followed by DyLight594- DEAD Fixable Aqua (Invitrogen). For staining of DC subsets, conjugated goat anti-hamster IgG (Biolegend). The cover- LN cells were prepared by digesting LNs with 200 U/ml type slips were mounted using ProLong Gold Antifade mountant VIII collagenase (Sigma–Aldrich). For intracellular with DAPI (Molecular Probes, Eugene, OR, USA). Images staining, cells were cultured in the presence of 50 ng/ml PMA, were acquired using fluorescence microscopy, BZ-9000. 500 ng/ml ionomycin and 1 µM monensin for 5 h. Cells were washed, fixed with 4% paraformaldehyde (PFA), and per- meabilized with 0.1% saponin, followed by staining with anti- Isolation of epidermal cells from the skin IFN-γ, anti-IL-17 or anti-Foxp3 antibodies. For preparation of Shaved skin was cut out, and subcutaneous adipose tissues highly purified cells, cells were sorted by FACSAria II (Becton were removed by gently rubbing. After washing, the skin was Dickinson) or MoFlo XDP (Beckman Coulter, Miami, FL, USA). floated on 0.25 mg/ml thermolysin (Sigma–Aldrich) for 1 h at 37°C. Epidermis was peeled off, minced by scissors and dis- Quantitative reverse-transcription (RT)-PCR aggregated by vigorous pipetting. Epidermal cells were iso- RNAs from tissues and cells were purified using the Sepasol- lated by a cell sorter. RNA I Super (Nacalai Tesque, Kyoto, Japan) and GenElute mammalian total RNA miniprep kit (Sigma–Aldrich), respec- Generation of bone marrow-derived DCs and tively. Resulting RNAs were reverse-transcribed using the high macrophages capacity cDNA reverse transcription kit (Applied Biosystems, For generation of bone marrow (BM)-derived DCs (BMDCs), Foster City, CA, USA). For semi-quantitative analysis, synthe- BM cells were cultured at 2 × 106 cells/10 ml RPMI1640 sup- sized cDNA was amplified by PCR with Ex Taq DNA polymer- plemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml ase (TaKaRa, Kyoto, Japan) and a primer set (Supplementary streptomycin, 1% 2-mercaptoethanol and 20 ng/ml recom- Table 2 is available at International Immunology Online). For binant mouse GM-CSF (Peprotech, Rocky Hill, NJ, USA) in 172 CCR8 suppresses contact dermatitis 100-mm non-treated dishes. On day 3, 10 ml of the same detected in the culture supernatants from Ccr8−/− LN cell cul- fresh medium was added to the dishes. On day 6 and 8, ture (Fig. 2D). The results suggest that T cell priming against 5 ml of the medium was replaced with fresh medium, which DNFB is enhanced in Ccr8−/− mice. contained 10 ng/ml GM-CSF. On day 10, non-adherent cells were collected as BMDCs. For generation of BM-derived T cells from DNFB-primed Ccr8−/− mice induce enhanced macrophages (BMMPs), BM cells were cultured at 5 × 107 CHS responses in WT mice cells/10 ml RPMI1640 supplemented with 10% FBS, 100 U/ We examined T cell function of Ccr8−/− mice by employing ml penicillin, 100 µg/ml streptomycin, 1% 2-mercaptoethanol adoptive T cell transfer techniques. T cells were purified from and 20 ng/ml recombinant mouse M-CSF (R&D systems) in spleens and LNs, and transferred into recipient SCID mice 100-mm non-treated dishes. On day 3, 5 ml of the same fresh (Fig. 3A, left). After 24 h, CHS responses were induced in medium was added to the dishes, and on day 7, adherent these mice. We found that earlobe swelling was compara- cells were collected by a cell scraper with 2.5 mM EDTA. ble between WT T cell- and Ccr8−/− T cell-transferred mice (Fig. 3A, right), indicating that CCR8 deficiency in T cells Statistics does not affect the development of contact dermatitis. Two-tailed unpaired Student’s t-test was used for statistical Next, T cells were purified from dLNs ofCcr8 −/− mice evaluation of all experiments. P value < 0.05 was consid- 5 days after sensitization, and were intravenously transferred ered as significant; * <0.05; ** <0.01; *** <0.001. Data are into WT mice. Those mice were then challenged with DNFB expressed as a mean ± SD. (Fig. 3B, left). CHS development was aggravated in Ccr8−/− T cell-transferred mice compared with WT T cell-transferred Results mice (Fig. 3B, right), indicating that T cell priming is promoted in Ccr8−/− mice. Ccr8−/− mice are highly susceptible to DNFB-induced CHS We first investigatedCcr8 mRNA expression in DNFB- The migration of cutaneous DCs to dLNs is enhanced in induced CHS. Consistent with the observations in ACD Ccr8−/−mice patients (21), an increase of Ccr8 transcript was observed Because adoptive T cell-transfer experiments suggested that in sensitized skin compared with untreated skin (Fig. 1A). cutaneous DCs, but not T cells, affect priming responses, we To investigate the pathological role of CCR8 in contact der- analysed the DC function in Ccr8−/− mice in CHS. We exam- −/− matitis, we generated Ccr8 mice on the BALB/cA back- ined the expression of CCR8 in highly purified DC subsets, ground (Supplementary Figure 1 is available at International which were separated according to Ruedl et al. (39). (purity Immunology Online), and examined the development of CHS. > 99 %; Supplementary Figure 2 is available at International When CHS was induced in the earlobe by challenging with Immunology Online for the gating protocol). These DC sub- −/− DNFB, Ccr8 mice developed severer inflammation, as indi- sets corresponded to well documented LN DC populations cated by significantly increased punched earlobe weight at (35, 39, 40). We found that Ccr8 mRNA was significantly −/− 24 h (Fig. 1B). The extent of ear thickness in Ccr8 mice was expressed in DC subsets, including cDCs (CD11chiCD40int), significantly increased at 24 h after immunization (Fig. 1C). dDCs (CD11cintCD40hi), LCs (CD11chiCD40hi) and iDCs Histological analysis showed notable swelling of the earlobe (CD11cintCD40int), as well as in T and B cells, but not in and marked accumulation of granulocytes (Fig. 1D). Analysis BMLCs and BMDCs (Fig. 4A and Supplementary Figure S3 is of cytokine gene expression revealed that the expression of available at International Immunology Online). −/− the Il1b gene was significantly enhanced in Ccr8 mouse Because migration of cutaneous DCs into dLNs is a pre- ears compared with WT mouse ears (Fig. 1E), while Tnf and requisite to sensitize T cells to induce allergic responses, we Il10 gene expression was not. Although IgG antibody levels analysed the migration of these DC subpopulations after CHS −/− specific to DNP tended to increase inCcr8 mice compared induction. Significantly enhanced accumulation of dDCs, LCs with WT mice, the difference was not statistically significant, and iDCs was observed in the dLNs of Ccr8−/− mice com- consistent with our previous report that antibody levels don’t pared with WT mice (Fig. 4B and C), whereas the propor- affect the development of CHS [Fig. 1F (36)]. tions of plasmacytoid DCs (pDCs, CD11c+B220+) as well as T and B were similar between WT and Ccr8−/− −/− The T cell proliferating response is enhanced in Ccr8 mice mice (Supplementary Figure S4 is available at International Because the Ccr8 gene is highly expressed in T cells (37, Immunology Online). In contrast, no significant accumula- 38), we examined the proliferating response of Ccr8−/− LN tion of these DC subsets was observed under physiological cells. After sensitization of mice with DNFB, dLN cells were conditions without allergen challenge (Supplementary Figure isolated, and the proliferative response to DNBS was eval- S5 is available at International Immunology Online). High uated by [3H]-thymidine incorporation. The proliferative expression of MHC-II and CD86, activation markers for DCs, response of Ccr8−/− LN cells was significantly higher than that was similarly observed among WT and Ccr8−/− DC subsets of WT LN cells, while PMA/ionomycin-induced or anti-CD3- (Fig. 4D and E). The expression of CCR7, which is required induced proliferation was comparable (Fig. 2A and B). Under for the migration of DCs to dLNs through lymphatic vessels −/− the experimental conditions, Th1, Tc1 and Th17 cell popula- (6, 35), was also similar in these DCs from WT and Ccr8 tions were significantly expanded inCcr8 −/− mouse LNs mice. These results suggest that the migration of cutaneous after sensitization with DNFB compared with WT mouse LNs DCs into dLNs is increased at the sensitization phase of CHS (Fig. 2C). Higher concentrations of IFN-γ and IL-17 were also in Ccr8−/− mice. CCR8 suppresses contact dermatitis 173 A B C 12 200 *** * 190 (mg) *** ** 10 180 ) Vehicle DNFB 170 (% elling 8 160 Ccr8

6 elling 150 140 of ear sw Gapdh 4 130 Ear sw WT 2 120 110 Ccr8-/- Increment 0 100 -/- WT Ccr8 0012324 48 72 D Time (h) after 2nd immunization Vehicle (X 40) DNFB (X 40) DNFB (X 100)

WT

Ccr8-/-

E 2 * F ) ) 0.4

(/Hprt 1.5 0.3 (OD415

1 WTWT pression 0.2 -/-

ex Ccr8-/Ccr8- -DNP-Ab

0.5 anti 0.1 Relative

0 Serum 0 TnTnfIl1bIl10 WT Ccr8-/-

Fig. 1. DNFB-induced CHS is augmented in Ccr8−/− mice. (A) One day after the second immunization with DNFB or vehicle, the expression levels of Ccr8 mRNA in the skin of a WT mouse was analysed by semi-quantitative RT–PCR. The data were reproducible in another mouse. (B) Mice were sensitized on their abdomen with DNFB, and 5 days later, mice were challenged with DNFB on the ears. Twenty four hours later, earlobe swelling was assessed by the weight of punched earlobes (n = 11–12). The data were reproduced in another independent experiment. (C) Ear swelling was quantified by measurements of ear thickness using a caliper n( = 6–7), and is shown as percentage of thickness compared with ears at day 0. (D) Earlobe sections (5 µm) were stained with HE, and histology was examined by microscopy. (E) Cytokine gene expression in the ears from DNFB-immunized mice was analysed by quantitative PCR (n = 6–7). (F) Serum antibody titers specific for DNP were analysed by ELISA (n = 11–12). The data were reproduced in another independent experiment (F). Means ± SD are shown (B, C, E and F). *P < 0.05; **P < 0.01; ***P < 0.001 (two-tailed unpaired Student’s t-test).

Next, we measured the number of CD207+ DCs in the DNFB (Fig. 5A, left). Notably, this reduction was more sig- epidermis using fluorescence microscopy. The number nificantly observed inCcr8 −/− mice (Fig. 5A, right). These of epidermal DCs was reduced after sensitization with observations support the notion that Ccr8−/− DCs migrate 174 CCR8 suppresses contact dermatitis

Fig. 2. T cell sensitization against DNFB is enhanced in Ccr8−/− mice. (A) Five days after sensitization with DNFB, dLN cells were re-stimulated with DNBS. Recall proliferation of LN cells was determined by [3H]-thymidine incorporation (n = 3 each). PMA/ionomycin simulation was used as a control (n = 3 each). The data are representative of three independent experiments. *P < 0.05 (two-tailed unpaired Student’s t-test) (B) T cells were isolated from spleen and LNs, and were stimulated with plate-bound anti-CD3 antibody for 3 days. The proliferative response was determined by [3H]-thymidine incorporation. The data are presented as mean ± SD of triplicate wells, and representative of two independent + + experiments. (C) The proportion of Th1, Tc1 and Th17 cells in dLNs after antigen re-stimulation was analysed by flow cytometry. CD4 and CD8 T cells were stained with anti-IFN-γ or anti-IL-17. Mean ± SD of three independent experiments (n = 9 each). *P < 0.05; **P < 0.01 (two-tailed unpaired Student’s t-test). (D) The concentrations of IFN-γ and IL-17 in LN cell culture supernatants were determined by ELISA. Mean ± SD of three independent experiments (n = 9 each). **P < 0.01 (two-tailed unpaired Student’s t-test). CCR8 suppresses contact dermatitis 175

A WT T cells SCID mice Day -1 056 Ccr8-/- T cells SCID mice 5 (mg) Transfer SensitizationChallenge Measure 4 elling WT T cells 3 SCID SCID 2 of ear sw Ccr8-/- 1

Increment 0 1x1 x10 10^55 101x 10x 10^65 B Day0 Day5 6 hr 24 hr 4 * (mg) 3 elling

WT WT T cells sw WT 2 of ear

Ccr8-/- Ccr8-/- 1 Increment 0 -/- WTWT Ccr8-/Ccr8 -

Fig. 3. T-cell sensitization upon treatment with DNFB was enhanced in Ccr8−/− mice compared with WT mice. (A) Experimental protocol for T cell transfer to SCID mice is shown in the left. T cells were isolated from spleens and LNs of Ccr8−/− and WT mice by MACS, and transferred into recipient SCID mice (1 × 105 or 1 × 106 cells/mouse). After 24 h, these mice were treated with DNFB to induce CHS. Earlobe swelling was assessed by weighing punched earlobes (n = 9–10) (right). The data were reproduced in another independent experiment. (B) Experimental protocol for adoptive transfer of DNFB-sensitized T cells (left). T cells were purified from dLNs of DNFB-sensitized mice, and were intravenously transferred into recipient WT mice. After 6 h, these mice were challenged with DNFB. The increment of earlobe swelling was assessed after 24 h (n = 4–5) (right). Mean ± SD. *P < 0.05 (two-tailed unpaired Student’s t-test). more easily from the epidermal compartment to dLNs after KCs (Fig. 7A). We further compared Ccl8 expression in the sensitization. skin between steady and inflammatory states.Ccl8 transcript Then, we directly examined migration of cutaneous Ccr8−/− was elevated in DNFB-challenged skin (Fig. 7B), suggest- DCs after sensitization with FITC. The proportions of FITC- ing that CCL8 expression is enhanced during inflammation labeled dDCs, LCs and iDCs were significantly increased in the skin. in dLNs of Ccr8−/− mice (Fig. 5B and C). Immunofluorescent staining analysis revealed that FITC+CD11c+ cells were local- Discussion ized at the cortical area in dLNs, but not at the subcapsular Chemokines mediate the trafficking of leukocytes to inflam- sinus (SCS) (Fig. 6), consistent with previous reports (41, 42). matory sites and specific lymphatic organs (44), while adhe- We also observed that the proportion of Ccr8−/− FITC+CD11c+ sion molecules are important for tethering cells to a tissue cells tended to be higher in that region. These results indicate (45). In the skin, LCs are anchored to the epidermis through that CCR8 deficiency promotes migration of DCs from skin E-cadherin bonds to KCs under steady state conditions (46, to dLNs. 47). Upon antigen stimulation, LCs are activated to express CCR7 and migrate into local dLNs where CCL19 and CCL21 CCL8 expression is upregulated in the skin during the are expressed. CCL8 is also highly expressed in LCs and sensitization phase KCs in the skin. We showed in this report that the expression Both CCL8 and CCL1 are the ligands for CCR8. CCL8 is of CCR8 on LCs and CCL8 in KCs of the skin was upregu- expressed in the skin (31), whereas CCL1 is not (43). Thus, lated when the mice were cutaneously sensitized with DNFB, we hypothesized that CCL8 is important for the recruitment suggesting that the CCR8–CCL8 interaction is important for and tethering of CCR8+ DCs in the skin. Then, we exam- the migration and recruitment of inflammatory cells to the ined the expression of Ccl8 in cutaneous and immune cells. antigen stimulated skin. Nonetheless, we showed that Ccr8−/− Epidermal LCs (CD45+CD11b+) and KCs (CD45−CD11b−TC mice are more susceptible to DNFB-induced CHS, suggest- Rγ/δ−CD117−) were highly purified by flow cytometry (>95% ing that CCR8 rather negatively regulates CHS responses. pure), and the expression of Ccl8 was analysed by quantita- Accumulation of DCs including LCs, dDCs and iDCs in the tive PCR. Ccl8 was expressed in both epidermal LCs and dLNs was significantly enhanced inCcr8 −/− mice compared 176 CCR8 suppresses contact dermatitis

A B WT Ccr8-/- 7 0.004 0.008 0.015 (LC) 6 WT 0.02 (LC) (dDC) (dDC) 5 Ccr8-/-

Actb 4 / 3 0.047 0.047 Ccr8 2 0.268 (cDC) 0.347 (cDC) 1 (iDC) (iDC) 0 cDCs dDCs LCs iDCs C CD40 0.04 0.016 CD11c 0.6 0.08

* *** ) *

0.03 0.012 cells) 0.06 cells) 0.4 0.02 0.008 0.04 (% in LN cells) (% in LN (% in LN cells

(% in LN 0.2 0.01 0.004 0.02 LCs iDCs cDCs dDCs 0 0 0 0 * 4 4 5 5 1.E+0410 * 1.E+0410 1.E+0510 1.E+0510

* 1.E+04104 1.E+04104 1.E+03103 1.E+03103 1.E+033 1.E+033 (number ) 10 (number ) 10 (number) (number) 1.E+02102 1.E+02102 2 2 LCs

iDCs 1.E+02 1.E+02 cDCs dDCs 10 10

1.E+0110 1.E+0110 1.E+0110 1.E+0110 WT Ccr8-/-Ccr8-/- WT Ccr8Ccr8-/--/- WT Ccr8-/-Ccr8-/- WT Ccr8-/-Ccr8-/-

D dDCs LCs iDCs E dDCs LCs iDCs 15000 25000 10000

) 20000 8000

CD86 FI 10000 15000 6000 (M 5000 10000 4000 5000 2000 CD86

x 0 0 0

Ma 20000 25000 800 I-Ad of 15000 20000 600 % FI) 15000 10000 400 (M 10000 d

A 5000 200

I- 5000 0 0 0 CCR7 3000 2500 400 2000 300 FI) 2000 1500 Fluorescence Intensity 200 1000 1000 100 WT Isotype control 500 CCR7 (M 0 0 0 Ccr8-/- WT Ccr8-/- WT Ccr8-/- WT Ccr8-/-

Fig. 4. Accumulation of dDCs, LCs and iDCs is enhanced in Ccr8−/− dLNs at the sensitization phase. (A) DC subsets were highly purified from dLNs by a cell sorter (99% pure). The gating protocol is described in Supplementary Figure S2, available at International Immunology Online. The expression of the Ccr8 gene in DC subsets was analysed by real-time PCR. (B) After 24 h sensitization with DNFB, cells were isolated from dLNs, and DC subsets were analysed by flow cytometry. The data are shown in (C) (upper panel, percentage; lower panel, number;n = 5 each). The expression of CD86, I-Ad and CCR7 of each population was analysed by flow cytometry. A representative histogram and the MFI levels of DC markers are shown in (D) and (E), respectively (n = 5 each). The data are representative of three independent experiments. Means ± SD are shown (C and E). *P < 0.05; ***P < 0.001 (two-tailed unpaired Student’s t-test). with WT mice after sensitization. Furthermore, the number WT DCs. Consistent with this, we showed that migration of of DCs in the skin significantly reduced in DNFB-sensitized antigen-captured dDCs, LCs and iDCs from the skin to dLN Ccr8−/− mice, suggesting that cutaneous Ccr8−/− DCs eas- was significantly enhanced inCcr8 −/− mice upon sensitiza- ily migrated from the sensitized skin to dLNs compared with tion with FITC. Although CCR8 signaling induces Ca2+ influx CCR8 suppresses contact dermatitis 177

Steady Sensitization

A )

2 900 800 mm 700

WT in (/ * 600

l sk 500

ma 400 300 200 in epider 100 Ccr8-/-

LCs 0 WTWT Ccr8-/Ccr8-/--WWTTCCcr8cr8-/--/-

Steady Sensitization

B dDC (CD40hiCD11cint)iLC (CD40hiCD11chi) DC (CD40intCD11cint)

0.05

WT 0.03 0.11

0.10

Ccr8-/- 0.09 0.15 CD40 FITC C 0.2 0.25 *** 0.2 *** *** 0.2 0.15 0.15 0.15 0.1 0.1 (% of live cells) (% of live cells) (% of live cells) 0.1 LCs iDCs dDCs +

0.05 + 0.05

+ 0.05 FITC FITC FITC 0 0 0 WT Ccr8-/Ccr8-/-- WT Ccr8Ccr8-/-/-- WT Ccr8-/Ccr8-/-- 1.E+06106 *** 1.E+06106 ** 1.E+06106 ** ) ) 1.E+05105 1.E+05105 1.E+05105

4 4 4 (number ) 1.E+04 1.E+04 1.E+04 10 10 (number 10 (number

3 LCs 3 3

1.E+03 1.E+03 iDCs 1.E+03 dDCs 10 10 10 + + + 1.E+022 1.E+022 1.E+022 10 FITC 10 10 FITC FITC

1.E+0110 1.E+0110 1.E+0110 WT Ccr8-/Ccr8-/-- WT Ccr8-/Ccr8-/-- WT Ccr8-/Ccr8-/--

Fig. 5. CCR8 regulates migration of cutaneous DCs during the sensitization phase. (A) Epidermal cell sheets were prepared from the earlobe skin after 24 h-sensitization with DNFB. The sheets were stained with a biotin-conjugated anti-CD207 monoclonal antibody, followed by DyLight549- streptavidin. Microscopic photographs were taken using fluorescence microscopy (left; original magnification: ×40), and DC numbers are shown in the right (n = 5 each). (B) Mice were sensitized by painting with FITC on the shaved back skin. Twenty-four hours later, cells were isolated from dLNs, and the contents of FITC+ DCs were analysed by flow cytometry n( = 13 each). The gating protocol is described in Supplementary Figure S2, available at International Immunology Online. The data are shown in (C) (upper panel, percentage; lower panel, number; n = 5 each). The results are reproduced in the two independent experiments. Mean ± SD (A and B). *P < 0.05; ***P < 0.001 (two-tailed unpaired Student’s t-test). 178 CCR8 suppresses contact dermatitis

WT Ccr8-/- SCS SCS

FITC

SCS SCS

CD11c

SCS SCS

DAPI

SCS SCS

Merge FITC CD11c DAPI

Fig. 6. Localization of migratory DCs in dLNs after treatment with FITC. Mice were sensitized with FITC. Frozen sections were prepared from dLNs of these mice, and stained with hamster anti-CD11c monoclonal antibody followed by DyLight594-anti-hamster antibody (Red). FITC appears green. Nuclei were stained with DAPI (blue). Images were acquired by fluorescence microscopy. Areas surrounded by dotted lines show the SCS. Original magnification, ×20. Arrows indicate FITC+CD11c+ cells (yellow). CCR8 suppresses contact dermatitis 179

A B 1.2 6 *

1 5

0.8 4 t Hprt / 0.6 3 Ccl8 0.4 Ccl8/Hpr 2

0.2 1

0 0 SteadyDNFB- SensitizDNFBatio+ n state state

Fig. 7. Ccl8 expression is up-regulated in the skin after sensitization. (A) The expression of the Ccl8 gene in cells was analysed by quantitative PCR. KCs, keratinocytes; LCs, Langerhans cells; MEFs, mouse embryonic fibroblasts; BMDC, bone marrow-derived dendritic cells; BMMPs, bone marrow-derived macrophages. (B) WT mice were sensitized with DNFB on the skin. After 1 day, sensitized skin regions were isolated. Total RNA was extracted, reverse-transcribed and amplified by quantitative PCR. The data from two independent experiments were combined (n = 10 each). Mean ± SD are shown. *P < 0.05 (two-tailed unpaired Student’s t-test). and cell activation (23, 24, 48), we did not find any differ- induced, the migration of cutaneous DCs is strongly restricted ence in the expression of the maturation markers, MHC-II by the excess expression of CCL8 in the skin. and CD86, on DCs between WT and Ccr8−/− mice. These In conclusion, we have shown here that CCR8 is a nega- observations suggest that CCR8 expression on APCs is sup- tive regulator of APC migration from the skin to dLNs in CHS. pressive rather than promotive in the development of CHS This regulation of APC migration may be beneficial for the responses by negatively regulating migration of cutaneous hosts by keeping APCs in epidermis and preventing excess LCs, dDCs and iDCs from skin to dLNs upon stimulation with hapten-induced hypersensitivity (Supplementary Figure S7 is haptens. available at International Immunology Online).

It was reported that CCR8 is primarily expressed in Th2 cells (28) and plays an important role in the development of Supplementary data atopic dermatitis by recruiting Th2 cells into allergen-inflamed skin. CCR8 is also expressed in human and murine Treg cells Supplementary data are available at International Immunology (49–51), which suppress the development of T-cell-mediated Online. skin diseases (52). Therefore, we initially speculated that Ccr8−/− mice are highly susceptible to CHS, because Treg cell recruitment to inflammatory sites is impaired in the elici- Funding tation stage. However, we found that the sensitivity to CHS CREST of the Japan Science and Technology Agency (to Y. I.); was comparable between WT T cell-transferred and Ccr8−/− T the Science and Technology Research Promotion Program cell-transferred SCID mice. We also observed that the Treg for Agriculture, Forestry, Fisheries and Food Industry (to cell population was unchanged in the dLN between WT and Y. I.); Grand-in-Aid for Scientific Research from the Ministry of Ccr8−/− mice after immunization with DNFB (Supplementary Education, Science and Culture of Japan (24220011 to Y. I.); Figure S6 is available at International Immunology Online). and Grant-in-Aid for JSPS Fellows (11J09956 to R. Y.). These results suggest that T cells are not responsible for the exacerbation of CHS in Ccr8−/− mice. Conflict of interest statement: The authors declared no conflict of CCR8 contributes to trafficking of -derived DCs interests. (MDDCs) from skin to LNs. In an earlier study, Qu et al. (53) showed that the migration of latex-beads+ Ccr8−/− MDDCs to References the SCS of the LNs was reduced compared with WT MDDCs. 1 Holness, D. L. 2013. Recent advances in occupational dermatitis. In contrast, we found that iDC accumulation in dLNs was Curr. Opin. Allergy Clin. Immunol. 13:145. significantly enhanced inCcr8 −/− mice at the sensitization 2 Thyssen, J. P., Linneberg, A., Menné, T. and Johansen, J. D. phase. This apparent discrepancy could be explained by 2007. The epidemiology of contact allergy in the general pop- the difference of the models; we examined migration under ulation—prevalence and main findings.Contact Dermatitis 57:287. inflammatory conditions, whereas Quet al. did not induce 3 Förster, R., Schubel, A., Breitfeld, D. et al. 1999. CCR7 coordi- inflammation. We found thatCcl8 expression was upregu- nates the primary immune response by establishing functional lated in the skin after DNFB-sensitization. Thus, under the microenvironments in secondary lymphoid organs. Cell 99:23. physiological conditions, tethering of latex-bead-labeled 4 Saeki, H., Moore, A. M., Brown, M. J. and Hwang, S. T. 1999. Cutting edge: secondary lymphoid-tissue chemokine (SLC) and MDDCs by CCL8 in the skin may not be so strong, and CCL1, CC chemokine receptor 7 (CCR7) participate in the emigration which is expressed in the LN subcapsule, is involved in the pathway of mature dendritic cells from the skin to regional lymph recruitment of these cells to the dLN. In contrast, when CHS is nodes. J. Immunol. 162:2472. 180 CCR8 suppresses contact dermatitis

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