CXCL12-CXCR4 Engagement Is Required for Migration of Cutaneous Dendritic Cells
2007; Elsevier BV; Volume: 171; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2007.070225
ISSN1525-2191
AutoresKenji Kabashima, Noriko Shiraishi, Kazunari Sugita, Tomoko Mori, Ayako Onoue, Miwa Kobayashi, Jun‐ichi Sakabe, Ryutaro Yoshiki, Hirokazu Tamamura, Nobutaka Fujii, Kayo Inaba, Y. Tokura,
Tópico(s)Chemokine receptors and signaling
ResumoCCR7 is regarded as an essential chemokine receptor for cutaneous dendritic cell (DC) migration into the regional lymph nodes. However, complete migratory inhibition cannot be obtained in CCR7-deficient mice, suggesting that there exist other chemokine receptors involved in this process. Initially, we found that CXCR4 was highly expressed on migrated cutaneous DCs and that its ligand, CXCL12, was detected in the LYVE-1+ lymphatic vessels in the skin. FITC-induced cutaneous DC migration into the draining lymph nodes was impaired by the specific CXCR4 antagonist 4-F-Benzoyl-TN14003. Among FITC+ cells, Langerin+ Langerhans cells and Langerin− (dermal) dDC subsets were detected as CD11chigh+CD11bint+ cells and CD11chigh+CD11bhigh+ plus CD11clow+CD11bint+ cells, respectively, both of which were suppressed by CXCR4 antagonist. Moreover, in vivo contact hypersensitivity response was impaired by CXCR4 antagonist administered during the sensitization phase. The in vitro proliferative response to dinitrobenzene sulfonic acid of sensitized lymph node cells was inhibited by CXCR4 antagonist treatment. These findings demonstrated that CXCL12-CXCR4 engagement on cutaneous DCs plays a crucial role in the initiation of skin immune response by enhancing cutaneous DC migration. CCR7 is regarded as an essential chemokine receptor for cutaneous dendritic cell (DC) migration into the regional lymph nodes. However, complete migratory inhibition cannot be obtained in CCR7-deficient mice, suggesting that there exist other chemokine receptors involved in this process. Initially, we found that CXCR4 was highly expressed on migrated cutaneous DCs and that its ligand, CXCL12, was detected in the LYVE-1+ lymphatic vessels in the skin. FITC-induced cutaneous DC migration into the draining lymph nodes was impaired by the specific CXCR4 antagonist 4-F-Benzoyl-TN14003. Among FITC+ cells, Langerin+ Langerhans cells and Langerin− (dermal) dDC subsets were detected as CD11chigh+CD11bint+ cells and CD11chigh+CD11bhigh+ plus CD11clow+CD11bint+ cells, respectively, both of which were suppressed by CXCR4 antagonist. Moreover, in vivo contact hypersensitivity response was impaired by CXCR4 antagonist administered during the sensitization phase. The in vitro proliferative response to dinitrobenzene sulfonic acid of sensitized lymph node cells was inhibited by CXCR4 antagonist treatment. These findings demonstrated that CXCL12-CXCR4 engagement on cutaneous DCs plays a crucial role in the initiation of skin immune response by enhancing cutaneous DC migration. It is in the lymphoid organs that T lymphocytes and antigen-presenting cells such as dendritic cells (DCs) participate to generate adaptive immune responses.1Banchereau J Steinman RM Dendritic cells and the control of immunity.Nature. 1998; 392: 245-252Crossref PubMed Scopus (12349) Google Scholar, 2Cyster JG Chemokines and cell migration in secondary lymphoid organs.Science. 1999; 286: 2098-2102Crossref PubMed Scopus (859) Google Scholar, 3Cyster JG Chemokines and the homing of dendritic cells to the T cell areas of lymphoid organs.J Exp Med. 1999; 189: 447-450Crossref PubMed Scopus (279) Google Scholar There are two subsets of DCs in the skin, dermal DCs (dDCs) and epidermal Langerhans cells (LCs). The arrival of antigen-bearing DCs into lymph nodes from peripheral sites begins several hours after antigen exposure and reaches its peak for 1 to 3 days, depending on the type of antigen and DCs. However, the precise repertoire of signals that regulate these processes is not fully elucidated.2Cyster JG Chemokines and cell migration in secondary lymphoid organs.Science. 1999; 286: 2098-2102Crossref PubMed Scopus (859) Google Scholar, 3Cyster JG Chemokines and the homing of dendritic cells to the T cell areas of lymphoid organs.J Exp Med. 1999; 189: 447-450Crossref PubMed Scopus (279) Google Scholar, 4Sallusto F Lanzavecchia A Mobilizing dendritic cells for tolerance, priming, and chronic inflammation.J Exp Med. 1999; 189: 611-614Crossref PubMed Scopus (422) Google Scholar, 5Sallusto F Palermo B Lenig D Miettinen M Matikainen S Julkunen I Forster R Burgstahler R Lipp M Lanzavecchia A Distinct patterns and kinetics of chemokine production regulate dendritic cell function.Eur J Immunol. 1999; 29: 1617-1625Crossref PubMed Scopus (573) Google Scholar, 6Cyster JG Chemokines, sphingosine-1-phosphate, and cell migration in secondary lymphoid organs.Annu Rev Immunol. 2005; 23: 127-159Crossref PubMed Scopus (722) Google Scholar Recently, based on in vitro studies of chemotaxis and chemokine receptor expression5Sallusto F Palermo B Lenig D Miettinen M Matikainen S Julkunen I Forster R Burgstahler R Lipp M Lanzavecchia A Distinct patterns and kinetics of chemokine production regulate dendritic cell function.Eur J Immunol. 1999; 29: 1617-1625Crossref PubMed Scopus (573) Google Scholar, 7Vecchi A Massimiliano L Ramponi S Luini W Bernasconi S Bonecchi R Allavena P Parmentier M Mantovani A Sozzani S Differential responsiveness to constitutive vs. inducible chemokines of immature and mature mouse dendritic cells.J Leukoc Biol. 1999; 66: 489-494Crossref PubMed Scopus (134) Google Scholar, 8Sallusto F Schaerli P Loetscher P Schaniel C Lenig D Mackay CR Qin S Lanzavecchia A Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation.Eur J Immunol. 1998; 28: 2760-2769Crossref PubMed Scopus (966) Google Scholar and in vivo studies using relevant rodent models, central roles for various chemokines and their receptors in DC migration have been identified.2Cyster JG Chemokines and cell migration in secondary lymphoid organs.Science. 1999; 286: 2098-2102Crossref PubMed Scopus (859) Google Scholar, 3Cyster JG Chemokines and the homing of dendritic cells to the T cell areas of lymphoid organs.J Exp Med. 1999; 189: 447-450Crossref PubMed Scopus (279) Google Scholar, 4Sallusto F Lanzavecchia A Mobilizing dendritic cells for tolerance, priming, and chronic inflammation.J Exp Med. 1999; 189: 611-614Crossref PubMed Scopus (422) Google Scholar, 5Sallusto F Palermo B Lenig D Miettinen M Matikainen S Julkunen I Forster R Burgstahler R Lipp M Lanzavecchia A Distinct patterns and kinetics of chemokine production regulate dendritic cell function.Eur J Immunol. 1999; 29: 1617-1625Crossref PubMed Scopus (573) Google Scholar, 6Cyster JG Chemokines, sphingosine-1-phosphate, and cell migration in secondary lymphoid organs.Annu Rev Immunol. 2005; 23: 127-159Crossref PubMed Scopus (722) Google Scholar, 9Melchers F Rolink AG Schaniel C The role of chemokines in regulating cell migration during humoral immune responses.Cell. 1999; 99: 351-354Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar Using human monocyte-derived DCs, it was reported that immature DCs express CCR1, CCR2, CCR5, and CXCR1 and that the induction of DC maturation by lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), or CD40L results in up-regulated expression of CCR7 and CXCR4.8Sallusto F Schaerli P Loetscher P Schaniel C Lenig D Mackay CR Qin S Lanzavecchia A Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation.Eur J Immunol. 1998; 28: 2760-2769Crossref PubMed Scopus (966) Google Scholar CCR7 is a well-known chemokine receptor responsible for regulating DC function. CCR7 deficiency dramatically impairs migration of activated cutaneous DCs into draining lymph nodes 24 hours after fluorescein isothiocyanate (FITC) application, with profound morphological alterations in the architecture of secondary lymphoid organs.10Förster R Schubel A Breitfeld D Kremmer E Renner-Muller I Wolf E Lipp M CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs.Cell. 1999; 99: 23-33Abstract Full Text Full Text PDF PubMed Scopus (1932) Google Scholar However, it should be noted that this impairment of migration is incomplete. An another line of study using plt mice, which lack CCR7 ligands, has revealed that CCR7 ligand deficiency leads to an imperfect (approximately 70%) decrease in the number of FITC+ migrated DCs in the draining lymph nodes.11Gunn MD Kyuwa S Tam C Kakiuchi T Matsuzawa A Williams LT Nakano H Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization.J Exp Med. 1999; 189: 451-460Crossref PubMed Scopus (905) Google Scholar These data have suggested that there should exist other chemokines/chemokine receptors responsible for cutaneous DCs migration into lymph nodes. CXCR4 is a G-protein-coupled receptor expressed by a wide spectrum of cells. Its physiological importance in hematopoiesis and development of the vasculature and central nervous system has been emphasized by the lethal phenotype of its knockout mice. On the other hand, CXCR4 expression on monocyte-derived DCs is enhanced along with their activation, and DCs have chemotactic response to the CXCR4 ligand CXCL12 (stromal-cell derived factor-1) in vitro.12Saeki H Moore AM Brown MJ Hwang ST Cutting edge: secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes.J Immunol. 1999; 162: 2472-2475PubMed Google Scholar CXCR4 is also detected in human LCs, and its expression level is increased by granulocyte macrophage–colony-stimulating factor (GM-CSF).13Tchou I Misery L Sabido O Dezutter-Dambuyant C Bourlet T Moja P Hamzeh H Peguet-Navarro J Schmitt D Genin C Functional HIV CXCR4 coreceptor on human epithelial Langerhans cells and infection by HIV strain X4.J Leukoc Biol. 2001; 70: 313-321PubMed Google Scholar Nevertheless, there is little knowledge about the function of CXCR4 in cutaneous DCs and its contribution to directional migration of DCs on skin inflammation in vivo. CXCL12 is expressed by murine stromal cells in the red pulp of spleen and the medulla of lymph nodes and by human skin endothelial cells.5Sallusto F Palermo B Lenig D Miettinen M Matikainen S Julkunen I Forster R Burgstahler R Lipp M Lanzavecchia A Distinct patterns and kinetics of chemokine production regulate dendritic cell function.Eur J Immunol. 1999; 29: 1617-1625Crossref PubMed Scopus (573) Google Scholar, 14Pablos JL Amara A Bouloc A Santiago B Caruz A Galindo M Delaunay T Virelizier JL Arenzana-Seisdedos F Stromal-cell derived factor is expressed by dendritic cells and endothelium in human skin.Am J Pathol. 1999; 155: 1577-1586Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 15Hargreaves DC Hyman PL Lu TT Ngo VN Bidgol A Suzuki G Zou YR Littman DR Cyster JG A coordinated change in chemokine responsiveness guides plasma cell movements.J Exp Med. 2001; 194: 45-56Crossref PubMed Scopus (514) Google Scholar, 16Avniel S Arik Z Maly A Sagie A Basst HB Yahana MD Weiss ID Pal B Wald O Ad-El D Fujii N Arenzana-Seisdedos F Jung S Galun E Gur E Peled A Involvement of the CXCL12/CXCR4 pathway in the recovery of skin following burns.J Invest Dermatol. 2006; 126: 468-476Crossref PubMed Scopus (110) Google Scholar CXCL12/CXCR4 interactions are largely unique and nonpromiscuous. In mice, CXCL12 or CXCR4 gene knockouts generate a similar phenotype, characterized by deficient B lymphopoiesis and myelopoiesis and abnormal neuronal and cardiovascular development.17Nagasawa T Hirota S Tachibana K Takakura N Nishikawa S Kitamura Y Yoshida N Kikutani H Kishimoto T Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1.Nature. 1996; 382: 635-638Crossref PubMed Scopus (2020) Google Scholar, 18Zou YR Kottmann AH Kuroda M Taniuchi I Littman DR Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development.Nature. 1998; 393: 595-599Crossref PubMed Scopus (2134) Google Scholar, 19Ma Q Jones D Borghesani PR Segal RA Nagasawa T Kishimoto T Bronson RT Springer TA Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice.Proc Natl Acad Sci USA. 1998; 95: 9448-9453Crossref PubMed Scopus (1434) Google Scholar Embryonic lethality associated with either CXCR4 or CXCL12 gene knockouts emphasizes the critical and unique role played by these gene products during development. This chemokine also plays a critical role in lymphocytic circulation and immune surveillance in the postnatal life. In vitro, CXCL12 has potent chemoattractant properties for cells expressing CXCR4, such as monocytes, lymphocytes, and CD34+ hematopoietic stem cells. In light of the emerging significance of various members of the chemokine system in DC biology, we tested the hypothesis that the chemokine receptor CXCR4 and its ligand CXCL12 influence cutaneous DC function and adaptive immune responses. We found that CXCR4 is highly expressed on activated cutaneous DCs and that CXCL12 is expressed in the lymphatic vessels of the skin. Mice treated with CXCR4 antagonist exhibited significantly impaired cutaneous DC migration and reduced contact hypersensitivity (CHS) response. These findings collectively provide evidence for an important role of CXCR4 in cutaneous DC functions. Female C57BL/6 (B6) mice at 8 weeks of age were purchased from Japan SLC (Hamamatsu, Japan). Mice were maintained on a 12-hour light/dark cycle under specific pathogen-free conditions. Protocols were approved by the Institutional Animal Care and Use Committee of the University of Occupational and Environmental Health. For CXCR4 antagonist treatment, Alzet osmotic pumps (7-day duration, 0.5 μl per hour pumping rate; model 1007D; Durect Corporation, Cupertino, CA) were loaded with 40 mg/ml CXCR4 antagonist, 4F-benzoyl-TN14003,20Tamamura H Hiramatsu K Mizumoto M Ueda S Kusano S Terakubo S Akamatsu M Yamamoto N Trent JO Wang Z Peiper SC Nakashima H Otaka A Fujii N Enhancement of the T140-based pharmacophores leads to the development of more potent and bio-stable CXCR4 antagonists.Org Biomol Chem. 2003; 1: 3663-3669Crossref PubMed Scopus (96) Google Scholar, 21Tamamura H Hori A Kanzaki N Hiramatsu K Mizumoto M Nakashima H Yamamoto N Otaka A Fujii N T140 analogs as CXCR4 antagonists identified as anti-metastatic agents in the treatment of breast cancer.FEBS Lett. 2003; 550: 79-83Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar in saline and were implanted subcutaneously to the back under intraperitoneal anesthesia according to the manufacturer's instructions. The administered dose was calculated to be 0.48 mg per kg body weight per day. No toxicity of CXCR4 antagonist was observed at 5 μmol/L in vitro as reported previously.22Zannettino AC Farrugia AN Kortesidis A Manavis J To LB Martin SK Diamond P Tamamura H Lapidot T Fujii N Gronthos S Elevated serum levels of stromal-derived factor-1α are associated with increased osteoclast activity and osteolytic bone disease in multiple myeloma patients.Cancer Res. 2005; 65: 1700-1709Crossref PubMed Scopus (177) Google Scholar Moreover, the selectivity of the antagonist was confirmed by the finding that there was no significant inhibition against Ca2+ mobilization induced by MIP-1α stimulation through CCR5 (IC50 = 22 μmol/L) and against Ca2+ mobilization induced by sphingosine-1-phosphate stimulation through EDG3 (IC50 > 30 μmol/L) by the treatment of CXCR4 antagonist (data not shown). To characterize its specificity further, epidermal cell suspensions were applied to transwell for chemotaxis assay (see below for method). The chemotaxis of major histocompatibility complex (MHC) class II+ LCs to CXCL12 was inhibited by CXCR4 antagonist, but such inhibitory effect was not observed toward CCR7 ligand, CXCL21 (data not shown). Complete RPMI (cRPMI), RPMI 1640 medium (Sigma, St. Louis, MO) containing 10% heat-inactivated fetal calf serum (Invitrogen, Carlsbad, CA), 5 × 10−5 mol/L 2-mercaptoethanol, 2 mmol/L l-glutamine, 25 mmol/L HEPES (Cellgro, Herndon, VA), 1 mmol/L nonessential amino acids, 1 mmol/L sodium pyruvate, 100 units/ml penicillin, and 100 μg/ml streptomycin was used as culture medium. For depleting DCs, lymph node cells were dispersed and sorted to CD11c− population using CD11c microbeads with autoMACS per the manufacturers' protocol (Miltenyi Biotech, Gladbach, Germany). After depletion, the frequency of CD11c+ DC fraction was less than 0.02%. For organ culture assay, the skin of mouse ears were split along with cartilage, and the dorsal ear skin without cartilage was floated in a dermal side-down manner in 24-well tissue culture plates (Costar; Corning Life Sciences, Acton, MA) at 37°C. Twenty-four hours later, the cells in the wells were collected for analysis.23Kabashima K Sakata D Nagamachi M Miyachi Y Inaba K Narumiya S Prostaglandin E2-EP4 signaling initiates skin immune responses by promoting migration and maturation of Langerhans cells.Nat Med. 2003; 9: 744-749Crossref PubMed Scopus (259) Google Scholar Cell suspensions were prepared from lymph nodes by mechanical disruption on 70-μm nylon cell strainers (BD Falcon, San Jose, CA). For flow cytometry, cells were plated at a density of 1 × 106 cells per well in 96-well U-bottomed plates (Falcon). They were stained for 20 minutes on ice with antibodies (Abs) in 25 μl of phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS), 1 mmol/L ethylenediamine tetraacetic acid (EDTA), and 0.1% NaN3 and were washed twice with 200 μl of this buffer after each step. For staining with CXCR4, cells were preincubated with CD16/32 monoclonal Ab in 0.5% bovine serum albumin (BSA) containing RPMI 1640 medium for 30 minutes for resensitization and Fc receptor blocking and then stained as above. Data were collected on a FACSCanto or FACSCalibur (BD Biosciences, San Diego, CA) and analyzed with FlowJo software (TreeStar, San Carlos, CA). Abs used were as follows: phycoerythrin (PE)-conjugated anti-CXCR4 (2B11; BD Biosciences) and isotype-matched control IgG2a, PE-Cy5-conjugated anti-MHC class II Ab, PE-Cy7-conjugated CD11b and B220 Ab, and allophycocyanin (APC)-conjugated anti-CD11c Ab (all from BD Biosciences). Langerin was detected using a specific Ab (929F3; kindly provided by Sem Saeland, Schering Plough) in permeabilized cell suspensions, followed by visualization with anti-rat Ig conjugated to PE. For immunofluorescence analysis, the ears of B6 mice 24 hours after application with hapten were frozen in Tissue-Tek OCT compound 4583 (Sakura Finetechnical Co. Ltd., Tokyo, Japan). Cryostat sections (10 μm) were fixed in acetone and stained as described previously24Kabashima K Banks TA Ansel KM Lu TT Ware CF Cyster JG Intrinsic lymphotoxin-β receptor requirement for homeostasis of lymphoid tissue dendritic cells.Immunity. 2005; 22: 439-450Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar with the following reagents: goat anti-mouse CXCL12 Ab (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or rat anti-mouse LYVE-1 Ab (R&D Systems, Minneapolis, MN). Goat anti-CXCL12 Ab, after incubation with CXCL12 blocking peptide (62.5 μg/1 mg of antibody; Santa Cruz Biotechnology, Inc.) for 1 hour on ice and was centrifuged at 13,000 rpm for 1 minute, and the supernatant was used for control staining. Goat and rat Abs were detected using Alexa Fluor 488 rabbit anti-goat IgG (H+L) (Invitrogen, Molecular Probes, Carlsbad, CA) and PE-conjugated donkey anti-rat IgG (H+L) (Jackson ImmunoResearch, West Grove, PA), respectively, mounted with Prolong Gold antifade reagent (Invitrogen, Molecular Probes), and viewed with a Zeiss Axioplan fluorescence microscopy. Images were acquired on a 600CL-CU cooled charge-coupled device video camera (Pixera, Los Gatos, CA) and were processed with InStudio 1.0.0 (Pixera). Total mRNA was extracted from the mice ears with the SVTotal RNA Isolation system (Promega, Madison, WI) according to the manufacturer's protocol. Target gene expression was quantified in a two-step RT-PCR. cDNA was reverse transcribed from total RNA samples using the TaqMan RT reagents (Applied Biosystems, Foster City, CA). Murine CXCL12 (Assay ID: Mm00445552_m1) expression was quantified using TaqMan Gene Expression Assay (Applied Biosystems) in the ABI PRISM 7000 sequence detection system (Applied Biosystems). As an endogenous reference for these PCR quantification studies, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene expression was measured using the TaqMan rodent GAPDH control reagents (Applied Biosystems). The relative expression was calculated using the 2−ΔΔCT method.25Livak KJ Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔC(T) method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (127160) Google Scholar The expression of the target gene normalized to an endogenous reference and relative to calibrator is given by the formula 2−ΔΔCT. Gene expression in untreated mice was used as a calibrator expression to calculate ΔΔCT. Cells were tested for transmigration across uncoated 5-μm Transwell filters (Corning Costar Corp., Corning, NY) for 3 hours to CXCL12, CCL21 (R&D Systems), or medium in the upper or lower chamber and were enumerated by flow cytometry.26Ngo VN Tang HL Cyster JG Epstein-Barr virus-induced molecule 1 ligand chemokine is expressed by dendritic cells in lymphoid tissues and strongly attracts naive T cells and activated B cells.J Exp Med. 1998; 188: 181-191Crossref PubMed Scopus (400) Google Scholar For FITC-induced cutaneous DC migration, mice were painted on the shaved abdomen with 200 μl of 2% FITC (Sigma) dissolved in a 1:1 (v/v) acetone/dibutyl phthalate (Sigma) mixture, and the number of migrated cutaneous DCs into draining inguinal and axillary lymph nodes was enumerated by flow cytometry. In some experiments, mice ears were painted with 20 μl of 0.5% FITC, and draining cervical lymph nodes were analyzed as above. For CHS model, B6 mice were immunized by application of 25 μl of 0.5% DNFB in 4:1 (v/v) acetone/olive oil to their shaved abdomens on day 0. They were challenged on the right ear on day 5 with 20 μl of 0.3% (w/v) DNFB.27Kabashima K Murata T Tanaka H Matsuoka T Sakata D Yoshida N Katagiri K Kinashi T Tanaka T Miyasaka M Nagai H Ushikubi F Narumiya S Thromboxane A2 modulates interaction of dendritic cells and T cells and regulates acquired immunity.Nat Immunol. 2003; 4: 694-701Crossref PubMed Scopus (152) Google Scholar Ear thickness was measured before and 24 hours after challenge to assess inflammation. For treatment with CXCR4 antagonist 4F-benzoyl-TE14003, the compound was administered during the sensitization period (from 1 day before DNFB sensitization to 3 days after DNFB sensitization), elicitation period (from 1 day before challenge to 1 day after challenge), or both periods. For 2,4-dinitrobenzene sulfonic acid (DNBS)-dependent in vitro proliferation of lymph node cells, cells were prepared from draining axillary and inguinal lymph nodes 5 days after the DNFB sensitization on the abdomen. CXCR4 antagonist was implanted subcutaneously to the backside of the skin from 1 day before DNFB sensitization to 5 days after. Cells (4 × 105) were cultured for 3 days with DNBS (50 μg/ml), a water-soluble compound with the same antigenicity as DNFB, and were pulsed with 1 μCi of [3H]thymidine for the last 24 hours of culture. Data were analyzed using an unpaired two-tailed t-test. A P value of less than 0.05 was considered to be significant. Initially, we evaluated the expression levels of CXCR4 on migrated cutaneous DCs and resident DCs in the regional lymph nodes of mice by flow cytometry. FITC, known to induce DC maturation and mobilization,28Randolph GJ Inaba K Robbiani DF Steinman RM Muller WA Differentiation of phagocytic monocytes into lymph node dendritic cells in vivo.Immunity. 1999; 11: 753-761Abstract Full Text Full Text PDF PubMed Scopus (761) Google Scholar, 29Sato K Imai Y Irimura T Contribution of dermal macrophage trafficking in the sensitization phase of contact hypersensitivity.J Immunol. 1998; 161: 6835-6844PubMed Google Scholar was painted on the shaved abdomen, and the regional lymph node cells were isolated 24 hours later. After Fc receptor blocking with CD16/32 Ab (BD Biosciences) for 30 minutes, cells were incubated with PE-labeled CXCR4 or isotype-matched control Abs. Significant amounts of CXCR4 were detected in the MHC class II+ DCs, and among them, the FITC+ migrated cutaneous DC subset expressed a higher level of CXCR4 than the FITC− resident DC subset (Figure 1A). As a comparison, we monitored the expression level of CXCR4 in B220+ B cells where CXCR4 was also expressed (Figure 1B). It is worth noting that the level of CXCR4 expression on the migrated cutaneous DCs was comparable or even higher than that on B cells. Then, we performed a skin explant culture assay and analyzed the cells that migrated into the culture medium 24 hours after incubation. We found that MHC class II+ CD11c+ cutaneous DCs were already positive for CXCR4 (Figure 1C), suggesting the precedent up-regulation of CXCR4 on cutaneous DCs in the skin, where DCs are ready to migrate toward lymphatic vessels. To assess the chemotactic activity of resident and migrated cutaneous DCs to CXCL12, we prepared draining lymph node cells 24 hours after FITC application and applied them on chemotaxis assay using transwells. Both FITC− MHC class II+ resident DCs and FITC+ MHC class II+ migrated DCs showed chemotactic response to CXCL12 in a dose-dependent manner (Figure 2A). The response was more pronounced in FITC+ MHC class II+-migrated DCs (Figure 2A). As a positive control, the chemotaxis test of B220+ B cells to CXCL12 was simultaneously performed in parallel with DCs (Figure 2B). To evaluate whether CXCL12-CXCR4 interactions could serve as an optional backup to CCL21-CCR7 interactions or coordinated interplay between them, we examined the chemotactic activity of LCs to CXCL12 and CCL21. Epidermal cell suspensions were incubated in cRPMI for 24 hours and applied to transwells with or without CXCL12, CCL21, or both in combination of the upper and lower chambers. The migrated epidermal LCs were identified as MHC class II+ cells in the lower chamber. When CXCL12 or CCL21 was added to the lower chamber, LCs had a good chemotactic response to either of them, but the additional effect was not observed with CXCL12 or CCL21 combinatorially administered to the lower chamber (Figure 3). Interestingly, when CCL21 was added to the upper chamber, the chemotactic response to CXCL12 was significantly abrogated, but such an effect was not observed in the chemotaxis to CCL21 with CXCL12 added to the upper chamber (Figure 3). These data suggest that CXCL12-CXCR4 interactions can interplay coordinately with CCL21-CCR7 interactions and implicate that when CXCL12 and CCL21 coexist, LCs preferentially migrate into CCL21-producing sites. In addition, the finding that CCL21 added to the upper chamber abrogated the chemotactic response of LCs to CXCL12 and CXCL12 added to the upper chamber unaffected the response to CCL21 raised a possibility that CCL21 down-regulates the expression of CXCR4 and CXCL12 does not affect CCR7 expression. We thus stained MHC class II+ LC in the epidermal cell suspensions for CXCR4 and CCR7 5 hours after incubation with CCL21 or CXCL12. The treatments, however, did not alter the chemokine receptor expression levels at all (data not shown). It has been demonstrated that CXCL12 is expressed in the medullary cords of regional lymph nodes and human skin endothelial cells,14Pablos JL Amara A Bouloc A Santiago B Caruz A Galindo M Delaunay T Virelizier JL Arenzana-Seisdedos F Stromal-cell derived factor is expressed by dendritic cells and endothelium in human skin.Am J Pathol. 1999; 155: 1577-1586Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 15Hargreaves DC Hyman PL Lu TT Ngo VN Bidgol A Suzuki G Zou YR Littman DR Cyster JG A coordinated change in chemokine responsiveness guides plasma cell movements.J Exp Med. 2001; 194: 45-56Crossref PubMed Scopus (514) Google Scholar, 16Avniel S Arik Z Maly A Sagie A Basst HB Yahana MD Weiss ID Pal B Wald O Ad-El D Fujii N Arenzana-Seisdedos F Jung S Galun E Gur E Peled A Involvement of the CXCL12/CXCR4 pathway in the recovery of skin following burns.J Invest Dermatol. 2006; 126: 468-476Crossref PubMed Scopus (110) Google Scholar and its expression level is increased by skin wounding.16Avniel S Arik Z Maly A Sagie A Basst HB Yahana MD Weiss ID Pal B Wald O Ad-El D Fujii N Arenzana-Seisdedos F Jung S Galun E Gur E Peled A Involvement of the CXCL12/CXCR4 pathway in the recovery of skin following burns.J Invest Dermatol. 2006; 126: 468-476Crossref PubMed Scopus (110) Google Scholar However, the role of CXCL12 in the context of antigen exposure remains unknown. We performed an immunohistochemical analysis on CXCL12 expression in the mouse skin and detected a significant amount of CXCL12 signal in the dermis 24 hours after epicutaneous immunization with DNFB (Figure 4A, right top). In addition, we observed that the CXCL12-expressing cells were tightly associated with LYVE-1+ lymphatic vessels, whereas CXCL12-expressing cells were sparse in other areas of the skin (Figure 4A, right middle and bottom). The specificity of this staining was confirmed by the blocking peptide treatment or isotype-matched Ab staining (Figure 4A, left). On the other hand, the expression level of CXCL12 was less significant in the steady state (data not shown). We then examined CXCL12 mRNA levels in the skin of ears treated with 20 μl of 0.2% DNFB ears for 6, 12, 24, and 48 hours. The intensities of CXCL12 probes were normalized against GAPDH as an endogenous control. The amount of CXCL12 mRNA in the DNFB-treated skin was expressed as the mean relative to that in non-DNFB-treated skin using the ΔΔCT method. Its expression was induced 6, 12, 24, and 48 hours after hapten application with peak expression at 12 to 24 hours (
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