Journal of Cell Science uaeu C odann yp oe sasse yFT knpitn nmc.Fnly eso htdlto fteCX3CL1 the of deletion that show we Finally, mice. in painting skin FITC by assessed as nodes in lymph receptor draining to DCs cutaneous eia eerhCuclHmnImnlg nt etealIsiueo oeua eiie onRdlfeHsia,Haigo,Ofr OX3 Oxford Headington, Hospital, Radcliffe John Medicine, Molecular of Institute Weatherall Unit, Immunology Jackson Human Council G. Research David Medical and Johnson* A. lymphatics Louise inflamed through of cells trafficking dendritic promotes CX3CL1 chemokine The Article Research n erdcini n eimpoie htteoiia oki rpryattributed. properly is Attribution work Commons original the Creative that the provided of medium distribution terms any use, the in unrestricted reproduction under permits and distributed which article (http://creativecommons.org/licenses/by/3.0), Access License Open an is (Johnson This VCAM-1 leukocyte and of ICAM-1 Martı induction including by 2010; accompanied molecules, is Jackson, adhesion 2003) al., and mechanism uninflamed et (Johnson Fontecha activated in main in endothelium CCL21 least of lymphatic the production at increased when DCs, as inflammation, of (La proposed amoeboid entry tissue chemokine-directed, been lymphatic termed Indeed, controlling process has 1999). a al., cognate movement, et migration, its Saeki integrin-independent 2004; binding al., through et (Fo resting both CCR7 inflammation, during receptor vessels and lymphatic to conditions DCs required the of that is chemotaxis documented CCL21 for well chemokine is It endothelial-expressed steps afferent response. crucial lymphatic most immune into the an of initiating one tissue in is endothelium transmigration the lymphatic antigen- This the 2003). from across al., professional et migration (Steinman to capillaries and lymphatic phagocytes induce cells from cytokines inflammation, presenting DCs pro-inflammatory of of induction and maturation the endotoxins or bacterial infection draining al., Following et to Steinman 2002; 2003). al., surveillance tissues et immune (Scheinecker normal tolerance from peripheral maintain and to (DCs) required is cells nodes lymph dendritic of Trafficking Introduction words: Key monolayers recombinant HDLEC both that across time DCs first of the migration for basolateral-to-luminal show promote We CX3CL1 shed metalloproteinases. HDLECs secreted matrix molecule, vitro through endogenous adhesion surface and leukocyte basolateral a CX3CL1 their as soluble CX3CL1 at transmembrane CX3CL1 predominantly all express entry. both virtually vessel which endothelium, lymphatic lymphatic cells, lymphatic in inflamed endothelial roles across the in blood have However, induced unlike Transmigration pairs CCR7. is receptor chemokine-receptor (fractalkine) responses. additional its CX3CL1 and that chemokine immune CCL21 likely transmembrane TNF- afferent chemokine is the ongoing the it via that involve and report of periphery to obscure we known stimulation remain the Here, is transit from resulting and DC process of (DCs) a this details cells in precise with step dendritic first nodes, antigen-loaded the constitutes of lymph endothelium trafficking draining increased to by lymphatics characterised is inflammation Tissue Summary 10.1242/jcs.135343 doi: 5259–5270 126, ß Science Cell of Journal 2013 August 13 Accepted ( correspondence for *Author UK 9DS, hsetbihn rvosyurcgie oefrti tpclceoiei euaigD rfikn hog h lymphatics. the through trafficking DC regulating in chemokine atypical this for role unrecognised previously a establishing thus 03 ulse yTeCmayo ilgssLtd Biologists of Company The by Published 2013. utemr,w show we Furthermore, . a tetdhmndra ypai nohla el HLC)and (HDLECs) cells endothelial lymphatic dermal human -treated memn ta. 08.Hwvr pnteostof onset the upon However, 2008). al., et ¨mmermann Cx3cr1 ypai,Ceoie Ctafcig nlmain nohla cell Endothelial Inflammation, trafficking, DC Chemokine, Lymphatic, 2 rtre l,19;Gn ta. 99 Ohl 1999; al., et Gunn 1999; al., et ¨rster / 2 [email protected] C eut nmrel eae ypai trafficking lymphatic delayed markedly in results DCs nvivo in htnurlsn nioisaantC3L rmtclyrdc legnidcdtafcigof trafficking allergen-induced reduce dramatically CX3CL1 against antibodies neutralising that ) ´ n- namne htwsaprnl needn fitgi n Gi and integrin of independent apparently was that 1997). an manner leukocytes al., of a adhesion et in endothelial comprising shear-resistant (Bazan chemokine shown tight, originally induce stalk was acids, to CxxxC CX3CL1 mucin-like of form novel extended amino membrane-anchored This most an a 373 and Unlike containing integral motif I of domain respects. type large, extracellular several protein a as distinct in synthesised membrane structurally is is chemokines and CX3CL1 1997) chemokines, al., other et Pan from 1997; al., et endothelium vascular (Bazan activated and epithelium intestinal might neurons, that chemokines CX3CL1 upon further primarily focusing identify (fractalkine). entry, lymphatic to to sought contribute present sequential this we In a 2007). redundancy. either chemokine study, CCL21 multiple indicating or al., and action non-additive, of CXCL12 be mode et to of in lymphatic reported effects (LCs) (Kabashima were chemotactic cells for responses the Langerhans reported its Interestingly, and and hypersensitivity DCs been CXCR4 dermal for skin has both role in of a lymph (SDF-1) addition, CCR8 migration to In CXCL12 tissues use 2004). the al., ligand to from et migration (Qu shown during nodes been CCR7, to have lymphatics. addition the DCs conventional in trafficking Monocyte-derived leukocyte through controls that transmigrate chemokine can DCs 2006), mechanisms. integrin-dependent al., et XC1i xrse nanme fcl ye including types cell of number a in expressed is CX3CL1 only the not is CCL21 that clear increasingly becoming is It nvivo in nvivo in namuemdlo knhprestvt.However, hypersensitivity. skin of model mouse a in n mardtasypai migration translymphatic impaired and nvitro in nvitro in 5259 in in , Journal of Cell Science Results eefudt ert itei n XC1(i.1–) whereas 1A–C), (Fig. culture TNF- CX3CL1 any with tissue if stimulation little in secrete to HDLECs found Resting chemokine, were respectively. lysates, cell-associated detergent ELISA and and by supernatant assayed cells soluble and cytokines three pro-inflammatory for absence endothelial of or typically panel presence the a S1), lymphatic in of cultured were Fig. resection, dermal from material passages human (supplementary (HDLECs) passage endothelium, lymphatic in early CX3CL1 of expression investigate To cytokines after pro-inflammatory HDLECs with in stimulation induced dramatically is CX3CL1 CD14 including leukocytes, al., by expressed et Umehara remain 2000; al., inflammation et in Goda 2006; roles 2001). al., soluble et respective (Garton and their obscure The 2004). membrane-bound 2003). and al., al., between CX3CL1, et Hundhausen conventional differences et 2001; promote functional al., to Viemann et shown (Garton were 1997; chemotaxis metalloproteinases ADAM17 and and al., disintegrin by ADAM10 the generated et with CX3CL1 cleavage of Imai proteolytic forms Haskell soluble 1998; 1999; however, al., Subsequently et al., Fong 1997; al., et et (Bazan activation protein 5260 ahrta ebaeacoe hmkn rmtsDC endothelial promotes lymphatic lymphatic and chemokine by both soluble vessels transmigration, that lymphatic secreted membrane-anchored and towards actively migration inflammation lymphatic than to is regulating Here, response rather 2000). CX3CL1 in in al., cells et that receptor endothelial (Jung detail show its in we explored and not specific was CX3CL1 the trafficking function, of immune the in role Although defects established. apparent mice be no DC CX3CR1-knockout concluded to of constitutive of yet of control has characterisation the original lymph for extension However, the axis 2000). in CX3CL1–CX3CR1 in within al., migration promote the sensing et antigen of DCs (Muehlhoefer of significance to lumen purpose and/or Curiously, intestinal the 2012). for monocytes the shown propria, Greaves, by lamina intestinal also and dendrites White transepithelial was of 2007; al., 2001; CX3CL1 pathogenesis al., et et rheumatoid monocyte (Moatti supporting in damage Tacke by atherosclerosis, tissue seemingly exacerbating implicated and – as recruitment fibrosis renal been such and CX3CL1– arthritis leukocyte the diseases has supporting 2007), al., to inflammatory axis et addition (Rao In blood CX3CR1 al., 2000). the et from al., Imai extravasation et 2010; and al. Jung DCs et 1997; (Geissmann tissue-resident cells of Langerhans subsets epidermal and lineage DC macrophage omblsto fpepcae hmkn.Ti a supported CX3CL1 was This suppressed chemokine. pre-packaged completely opposed of as mobilisation agents regulation to transcriptional both with in C, consistent shown production, As and cycloheximide. inhibitor actinomycin 1B inhibitor synthesis Fig. polymerase protein RNA the the or not of compared D presence we data TNF- the with in regulation, stimulated and or of of when HDLECs in mode 1C degrees levels (Fig. observed the CX3CL1 Similar characterise HDLECs were To effect. detergent-lysed shown). CX3CL1 no in cell-associated had assessed upregulation of IL-6 modest IL-1 induction and more ng/ml) comparison, induced (15–30 (LPS) By lipopolysaccharide ng/ml). 100 h oercpo o XC1i XC1 hc swidely is which CX3CR1, is CX3CL1 for receptor sole The ora fCl cec 2 (22) 126 Science Cell of Journal nvitro in a nue rmtcices (typically increase dramatic a induced and nvivo in a IL-1 , . + el ftemonocyte- the of cells b IFN- , a ihralone either , c and XC1sann napooto fTNF- of intracellular proportion diffuse a however, in revealed staining Significantly, saponin CX3CL1 as 2A,B). with detached, (Fig. or permeabilisation monolayers suspensions adherent whether of as single-cell no regardless stained surface, Curiously, were cell the HDLECs antibodies. on detected labelled was CX3CL1 fluorescently with TNF- HDLECs, staining activated and in control CX3CL1 of intact induction confirm further To HDLECs activated of the from surface shed immediately is CX3CL1 Membrane-anchored Jhsne l,20) a lotecuieyluminal exclusively HDLECs Factor almost Willebrand von activated marker was the in with 2006), HDLECs induced permeabilised are al., ( which et secreted and of (RANTES) (Johnson CCL5 was both chemokines the CX3CL1 CCL2, of secretion contrast, Strikingly, By bottom were and ELISA. ( top predominantly inserts (i.e. by surfaces basolateral Transwell chambers) and polarised, luminal on both was TNF- from CX3CL1 with cultured of stimulated monolayers secretion whether HDLEC determine To monolayers basolateral HDLEC the of from surface predominantly shed that is in CX3CL1 involved suggesting HDLEC. sheddases from prime CX3CL1 3C), the of (Fig. ( reduced secretion are endogenous extent also ADAM17 GW280264X lesser I and ADAM10 a inhibitor and to MMP8 MMP2 the GI254023X although and addition, In shedding II 3B). CX3CL1 inhibitor (Fig. or control MMP9 a viability a and (MCP- as Importantly, CCL2 served HDLEC which chemokine S3). 1), cleavage-independent affected revealed secretion the Fig. of significantly material secretion ADAM17 CX3CL1 GW280264X inhibitor supplementary and neither of and and ADAM10 3A (Fig. inhibition TNF- the GI254023X of concentration-dependent with Incubation inhibitors 2003). monolayers cells 2000; al., al., HDLEC et stellate Goda et 2001; al., hepatic et Hundhausen cells Garton and 2009; endothelial al., activation- et fibroblasts vascular (Bourd-Boittin and from transfected constitutive CX3CL1 both (HUVECs), of mediate shedding to induced shown mediate MMP2 been that and have ADAM17 metalloproteinases ADAM10, the Previously, identify shedding. CX3CL1 to ADAM17 sought and ADAM10 we by Next, mediated is shedding CX3CL1 Fg 1D). abundant (Fig. detected which analyses, CX3CL1 PCR transcriptase reverse by niiulHLC n lordcdteaon ertdinto of secreted surface amount cell the the reduced at also and CX3CL1 HDLECs of 2A–C led individual Fig. accumulation inhibitor of in significant inclusion shown the to S2, As Fig. activity. material supplementary Ilomastat sheddase and inhibitor block metalloproteinase to matrix (GM6001) broad-range possibilities, the these of between distinguish and TNF- To membrane with plasma the stimulation rapidly. the to shed to targeted then traffics not it is is that protein or the membrane that plasma either indicating 2A), (Fig. h utr eim(i.2) hs eut niaeta the that almost indicate surface HDLEC synthesis. results the after from shed These immediately is CX3CL1 2D). of (Fig. form soluble medium culture the . 0)(i.4,) utemr,da-loecnesann of staining dual-fluorescence Furthermore, 4B,C). (Fig. 90%) RAfo TNF- from mRNA . 0)fo h aoaea ufc Fg 4A). (Fig. surface basolateral the from 70%) a a a eetdi h rsneo absence or presence the in repeated was a n hmkn erto measured secretion chemokine and siuae el eeiae after imaged were cells -stimulated a tetdbtntcnrlHDLECs control not but -treated a siuae cells -stimulated , a -stimulated 5)than 25%) Journal of Cell Science emaiiain(i.5.N XC1wsdtce within detected was CX3CL1 both TNF- No podoplanin from resting 5). and (Fig. sections frozen permeabilisation resected of endothelium, immunostaining freshly lymphatic intact performed in we expression CX3CL1 assess vessels lymphatic To inflamed in expressed is CX3CL1 blood suggest in findings (IL-8) These 1998). CXCL8 luminal al., as with et that (Wolff associated such cells organelles chemokines endothelial the Weibel–Palade of – within secretion 4D,E) CX3CL1 (Fig. no bodies essentially showed (vWF) emaiiaino h etos infcnl,n CX3CL1 no Significantly, after sections. only visible the were of that patches TNF- permeabilisation intracellular of in endothelium vessel skin, lymphatic treated in detected were levels ypodtsu Plrmne l,2001). al., are et that downstream (Palframan in CCL2 functions tissue remote lymphoid as for vessels such lymphatic would in chemokines This secreted from vessels. lymphatic CX3CL1 of surface distinguish inner to, the adjacent on, than directly rather events influencing perivascularly, functions nvivo in ypai nohla el(E)drvdCX3CL1 (LEC)-derived cell endothelial lymphatic , + ypaisi omlsi.B otat high contrast, By skin. normal in lymphatics a tetdhmndri after dermis human -treated nvivo in a - moiie hmkn Ptle l,20) nadto to of addition upregulation to In an than led 2001). rather also al., phase sensitisation fluid et CX3CL1 oxazolone a that as (Patel CX3CL1, fact acts chemokine the 2011; and with al., heparin immobilised et consistent bind (Tal and not CCL21 2013) does for be al., location could et this Little Weber of in panel). reports vessels, lymphatic to left of in contrast surfaces bottom subendothelial present lymphatic 6B, the be near (Fig. to detected lumen in inflamed appeared although vessel also pattern, amounts the perinuclear in residual a sections, in some CX3CL1 seen be could Furthermore, endothelium 6B). (Fig. n upeetr aeilFg 4 XC1sann was staining CX3CL1 S4, Fig. podoplanin in material detected supplementary and CX3CL1 mice, of and C57Bl/6 TNF- and investigated surface contact- explanted BALB/c in oxazolone both the we from within skin endothelium at hypersensitised lymphatic Next, podoplanin in induction with endothelium. colocalise lymphatic to appeared tiig ycnrs,C3L ol eraiydtce on detected readily be before could X-100 CX3CL1 Triton podoplanin contrast, with permeabilised By been staining. had tissue when XC1dpnetlmhtctafcig5261 trafficking lymphatic CX3CL1-dependent 2 rmusiuae cells. unstimulated are * from three case, of each experiment in one shown from data Representative control. a of Comparison (mean (D) ELISA by assessed with (CHX), hours cycloheximide 24 for stimulated and HDLECs TNF- (B) of supernatant (C) the lysates in detergent pro- and individual (A) with cytokines hours inflammatory 24 for stimulated HDLECs activated in cells. expression endothelial CX3CL1 lymphatic of Induction 1. Fig. tetd(4hus DEsb TPRwith RT-PCR by HDLECs hours) (24 -treated lo ailre nnnpreblsdtissue non-permeabilised in capillaries blood a ntepeec fatnmcnD(cD or (ActD) D actinomycin of presence the in a tetdmuedri.A hw nFg 6A Fig. in shown As dermis. mouse -treated + ypai esl nbt ae,btonly but cases, both in vessels lymphatic CX3CL1 P , .3i oprsnwt CX3CL1 with comparison in 0.03 RAlvl nrsigadTNF- and resting in levels mRNA eeso XC1scee from secreted CX3CL1 of Levels b 6 atna a as -actin s.e., n 5 3). Journal of Cell Science Fg C.W is esrdtecpct feoeosyadded exogenously CCR7 of capacity receptor the chemokine measured express first lymphoid We MDDCs 7C). essential in (Fig. the by 7A,B), mature (Fig. assessed to cytometry and flow CX3CR1, addition and receptor, analysis immature western CX3CL1 quantitative Both of levels monocyte- model. LPS significant with blood a treatment by human mature leukocyte as to using migratory induced (MDDCs) lymph, major DCs derived the afferent DCs, in we of chemotaxis possibility, population trafficking this leukocyte explore on directing To vessels. focused in is lymphatic role CX3CL1 to entry of a and/or secretion of polarised suggestive and clearly induction observed The across endothelium DCs lymphatic of transmigration polarised promotes CX3CL1 at a both retained surface, of than stored cell rather is part the molecule secretion, the for as that preparation confirm induced in and intracellularly inflammation humans, is and to mice CX3CL1 endothelium both that in lymphatic by time response first results pleiotropic These the pattern S5). for diffuse Fig. material more show (supplementary a tissue had the which within CCL2, podoplanin and around vessels, concentrated lymphatic were means the which are CCL20, data Representative and (D). TNF- CCL5 ELISA with by stimulated assessed saponin- also HDLECs in were and C CX3CL1 HDLECs and surface B control in cell in cells three. and levels from of intracellular supernatants CX3CL1 experiment in on surface CX3CL1 Ilomastat 630 cell of of Levels magnification: for Ilomastat. DAPI; effect (C) of with the values counterstained determine fluorescence were to Nuclei mean (red) respectively. HDLECs. derived monolayers, podoplanin activated HDLEC and of non-permeabilised (green) surface and CX3CL1 permeabilised the for from HDLECs CX3CL1 hours) of (24 shedding Rapid 2. Fig. 5262 ora fCl cec 2 (22) 126 Science Cell of Journal nvitro in and nvivo in . + A ulimnfursec tiigo oto n TNF- and control of staining Dual-immunofluorescence (A) hdigb ramn fHLC ihAA1 and antibody neutralising ADAM10 CX3CL1 in CX3CL1 with reduction the same as the of HDLECs to activated transmigration led of blockade MDDC Ilomastat, of or treatment inhibitors, face Importantly, ADAM17 of by basolateral polarity 4A). shedding the (Fig. observed the from HDLECs with secretion consistent CX3CL1 7F), luminal-to-basolateral (Fig. reverse not the did direction in chamber MDDCs upper of the migration to inhibit antibodies neutralising CX3CL1 of 7E, in by Fig. transit CX3CL1 MDDC HDLEC-expressed in inflammatory transmigration for mediating shown role DC As proposed the chamber. reduced confirming upper antibody the irrelevant to neutralising or added antibody neutralising IgG CX3CL1 rabbit a TNF- either using of presence assays out transmigration, carried DC we to of CX3CL1 contribution activation-induced the compared assess endogenous, transmigration to Next, of medium. of rate unsupplemented number with the the and both CX3CL1 increased MDDC of significantly transmigrating addition chamber 7D, upper Fig. in the shown to of As membranes 2006). FluoroBlok al., HDLECs Transwell et migration (Johnson of of under-surface monolayers the basolateral-to-luminal on across plated MDDCs stimulate labelled fluorescently to CX3CL1 a siuae DEs nthe in HDLECs, -stimulated a nvitro in ntepeec rabsence or presence the in 6 AShsorm B and (B) histograms FACS . 6 oal,addition Notably, . ..( s.e. a -stimulated n 5 )fo one from 3) , 50%, Journal of Cell Science ebaebudfr fC3L hti ciei mediating the in than active rather is secreted that the transmigration. CX3CL1 DC is of it form conclude membrane-bound we Hence, 6J). (Fig. MMP8 to * inhibitors three; with means of but the experiment B are from and Data shed A MMP2/9. CX3CL1 to and of conditions amount similar The under and (C) HDLECs GI254023X ELISA. inhibitors by ADAM10/17 assessed TNF- the GW280264X, of or presence the Ilomastat in either or with (control) alone and medium ADAM10 in cultured by cleavage involves CX3CL1 ADAM17. of Shedding 3. Fig. h muto XC1()adCL B hdfo HDLECs from shed (B) CCL2 and (A) CX3CL1 of amount The P , 0.03. 6 ..( s.e. n 5 )fo n representative one from 3) a o 4hours, 24 for xoe oete XC1o C2.C3L exposure CX3CL1 CCL21. or CX3CL1 MDDCs with the either reactivity selectively of compared to that and 2010). conformation 1992) exposed antibody al., Jackson, active et an (Dransfield and mAb24, an (Johnson used binds CCL21 we for Accordingly, documented chemokine-independent of that activation across involves transmigration a transmigrate DC in still in 2010). can Jackson, i.e. and DCs (Johnson manner endothelium, input G- pertussis when of lymphatic transmigration. by that 15–20% inhibited as shown are to toxin, have receptors additional studies chemokine contribute that protein-coupled previous chemokines and our Furthermore, sequentially endothelium unidentified act transmigration. CCL21 lymphatic yet and DC CX3CL1 activated of that in possible inhibition additive, is not complete results it was The antibodies Hence, than the Transwell. of of less effect the neutralising the yielding consequences of that CX3CL1 show chamber and 7K) the upper (Fig. CCL21 the investigated with in block antibodies we dual a 2010), imposing Jackson, and ietterbsltrlt-uia transmigration. basolateral-to-luminal TNF their activate direct from can that secreted time endothelium first CX3CL1 the for demonstrate soluble results time- These on hour S6). Fig. effect 4 the CCL21 material the significant of within of HDLECs mature no those in frame levels to had and VCAM-1 comparable or CX3CL1 immature ICAM-1 were However, both that 7G–I). (Fig. levels of to reactivity MDDCs, mAb24 increased xzln-yesniie idtp ie oallow To topical were of BMDCs wild-type skin mice. types, inflamed cell the the bone between wild-type discrimination of into migration co-injection oxazolone-hypersensitised the and after wild-type compared To from mice, lymphatics. (BMDCs) we the DCs within either question, marrow-derived in migration role subsequent this a or address play to must entry receptor initial its circulation. via and blood the exclusively CX3CL1 entering almost Hence, by than nodes rather lymph lymphatics, to afferent migrate DCs Cutaneous of migration DCs lymphatic impairs CX3CR1 of deletion Genetic of trafficking lymphatic in CX3CL1 of DCs DCs role of endogenous transmigration the in assessed role a next and LEC primary CX3CL1 in of expression inflammation-induced established Having mouse inflammation a skin in of DCs model of trafficking lymphatic promotes CX3CL1 C eoee rmdann yp oe falmc were shown). mice not all (data CX3CR1 of and nodes CCR7 both lymph express skin-derived draining to expected, found from As 8A,B). recovered (Fig. DCs controls IgG rabbit with etaiigatbde oCL1prilybokDC block partially CCL21 to TNF across transmigration antibodies neutralising fe 4hus lc fC3L upesdlmhnode lymph suppressed CX3CL1 of that FITC block showed of results trafficking hours, The 24 neutralising nodes. DCs after of lymph cytometry with cutaneous flow skin-draining by of measured disrupted was injected migration painting skin and FITC following CX3CL1, were against 6). antibodies (Fig. mice above mentioned Oxazolone-treated model skin-hypersensitivity induced ial,w vlae hte h oeo cino CX3CL1 of action of mode the whether evaluated we Finally, ie htatvtdHLC losceeCL1adthat and CCL21 secrete also HDLECs activated that Given XC1dpnetlmhtctafcig5263 trafficking lymphatic CX3CL1-dependent nvivo in nvitro in nvivo in + CD11c , sn h elcaatrsdoxazolone- well-characterised the using , rnmgainasy (supplementary assays transmigration a b tetdHDLEC -treated nernahso nDsand DCs in adhesion integrin 2 + nvivo in C by DCs a . siuae lymphatic -stimulated 0 hncompared when 70% b b nern iia to similar integrin, 2 nernsubunit integrin 2 nvitro in nvitro in Cx3cr1 (Johnson ,we 2 / 2 Journal of Cell Science niae yia nrclua oaino XC1 cl as 10 bars: Scale CX3CL1. of the location in intracellular hours typical 24 indicates for cultured TNF- or of (control) presence ( resected sections freshly frozen either permeabilised dermis, in (red) podoplanin TNF- and of vessels lymphatic in dermis. CX3CL1 human of Visualisation 5. Fig. 10 bar: TNF- Scale saponin-permeabilised TOPRO-3. in with (red) counterstained vWF mean and TNF- the HDLECs. (green) and are activated CX3CL1 control Data of of ELISA. surface surfaces by basolateral luminal assessed the and from basolateral secreted the selectively from is CX3CL1 4. Fig. 5264 ora fCl cec 2 (22) 126 Science Cell of Journal ulimnfursec tiigfrC3L (green) CX3CL1 for staining immunofluorescence Dual a uliwr onesandwt OR-.Arrow TOPRO-3. with counterstained were Nuclei . 6 ..( s.e. m .()Dgtlzo fidctdcl nD. in cell indicated of zoom Digital (E) m. n 5 )fo n ersnaieeprmn ftre * three; of experiment representative one from 3) , 4 m )o human of m) a siuae DE ooaesclue o 4huso loolkTaselisrs as inserts, Transwell Fluoroblok on hours 24 for cultured monolayers HDLEC -stimulated a a tetdHLC niae ako ooaiainwt eblPld ois uliwere Nuclei bodies. Weibel–Palade with colocalisation of lack indicates HDLECs -treated -activated m m. Cx3cr1 idtp MC (mean BMDCs wild-type eut eeas bandwe idtp and wild-type afferent when Similar obtained through apoptosis. also migration undergoing were than in rather results and delay vessels, hours a 24 lymphatic after experience CX3CR1-deficient nodes that indicating BMDCs identical, lymph almost were cervical hours 48 the BMDCs from knockout and recovered wild-type of numbers total the Importantly, yp oe eeldmre ifrne ntemigratory the cervical in excised differences the and marked however, wild-type of 9A, revealed properties Fig. in nodes shown material lymph As (supplementary EpCAM S7). marker Fig. lymph the DC showed and dermal (B7.2) migratory CD86 transfer. populations DC-selective molecule the co-stimulatory cell II, the adoptive class CD11c, MHC both of before expression injection, similar immediately before Importantly, Q-tracker525, with MC ess0.040 versus BMDCs htC3L ucin sa cesr hmkn o DC for chemokine accessory an lymph co- as the through than functions mobilisation rather time first CX3CL1 the animals, for indicate that findings separate These shown). into not (data injected injected were BMDCs and Q-tracker655 with labelled ipre y4 or,b ihreiigi h feetlmhor lymph efferent the in exiting either apoptosis by undergoing hours, largely were 48 but by hours, dispersed 24 within migrated had BMDCs wild-type aig4 or orahtesm yp oe [0.094 nodes lymph same the reach majority to the hours with 48 migrate, taking to slower significantly was population 2 / 2 mut fC3L A,CL B n C2()secreted (C) CCL2 and (B) CCL5 (A), CX3CL1 of Amounts MC t2 or;0.011 hours; 24 at BMDCs P , .3 D ulimnfursec tiigfor staining immunofluorescence Dual (D) 0.03. 6 nsitu in 0.006% Cx3cr1 ycnrs,the contrast, By . 6 Cx3cr1 Cx3cr1 nvivo in ... ess0.048 versus s.e.m.) 2 / 2 2 . 2 MC.Temjrt of majority The BMDCs. / / 2 2 MC eelabelled were BMDCs MC t4 hours). 48 at BMDCs 6 .0%wild-type 0.003% Cx3cr1 2 b Cx3cr1 / integrin 2 6 6 2 0.001% 0.018% BMDC 2 / 2 Journal of Cell Science iwaesoni ethn aes rhgnlveso ifrn esl in vessels different of views orthogonal panels; panels. left-hand right-hand in shown are view sdsrt el rlre lses(i.9) ept similar despite 9C), (Fig. clusters larger or cells either visible discrete injection, post as hours 24 at predominant heavily were fwl-yeand wild-type localisation between of differences obvious no to revealed trafficked nodes had lymph that DCs of Analysis cytometry. flow quantitative saoeadiae ycnoa irsoya a as microscopy confocal by imaged Triton and of immunostained presence above or respectively), as absence permeabilised, the and either (non-permeabilised in sections prepared X-100 Whole-mount were (B) skin shown. ear 100 inflamed are bars: of mice Scale four (green). from podoplanin images and Representative (red) and subjected CX3CL1 permeabilised mice for hypersensitivity, from stained skin skin oxazolone-stimulated ear topical inflamed and to skin ear contact- uninflamed of contralateral lymphatics dermal skin. in mouse CX3CL1 hypersensitised of Visualisation 6. Fig. 8husatraotv rnfr hscnimdtedlyin delay and the hours 24 confirmed at This 9C) transfer. (Fig. of adoptive site trafficking injection after skin hours the 48 allow and nodes to 9B) lymph respectively, draining (Fig. the Green both Tracker of imaging Cell microscopic fluorescent and Red Tracker Cell qa itrso idtp and wild-type of with mixtures equal intradermally injected were mice oxazolone-hypersensitised mgn ftesi neto iervae that revealed site injection skin the of imaging ofrhrivsiaeterl fC3L nD trafficking, DC in CX3CL1 of role the investigate further To Cx3cr1 Cx3cr1 2 / 2 2 A hl-on etoso control of sections Whole-mount (A) MC nlmhndsosre by observed nodes lymph in BMDCs / 2 ouain Fg B.B contrast, By 9B). (Fig. populations Cx3cr1 2 / 2 MC aeldwith labelled BMDCs Z sre.Snl lnsof planes Single -series. Cx3cr1 2 / 2 BMDCs m m. eew rsn vdneta XC1 h oemme fthe of member sole the CX3CL1, that evidence present we Here the Discussion also vessels. and the into capillaries DCs regulates lymphatic of CX3CL1 transit towards transendothelial that DCs subsequent conclude of we consistently migration Hence, a both fewer 9D). at of (Fig. and strikingly rate counterparts, underside hours, slower wild-type 24 their the lymphatic than of the endothelium across on course migrated mice CX3CR1-deficient plated the from BMDCs monolayers Over S8), (MDLEC) inserts. Fig. Transwell cell material endothelial (supplementary lymphatic dermal mouse hw opooeceoai fCX3CR1 been has of CX3CL1 chemotaxis of Haskell of form promote 1998; secreted to independent the al., shown contrast, et is By (Fong 1999). that al., involvement et protein mechanism Gi a and integrin in blood to receptor, endothelium binding its shear-resistant vascular confers with functional leukocytes that CX3CL1 on precise clear CX3CR1, is membrane-anchored the it and Although obscure, of still 1997). are interaction forms al., Hundhausen two ADAM17 2001; et the between the al., Pan differences et 2003; enzymes Garton by al., 1997; et conventional al., metalloprotease cleavage et a (Bazan controlled ADAM10 and and through protein disintegrin adhesion generated membrane chemokine, process. passive integral a lymphatics merely an as the past what in the of in complexity trafficking considered the cell to been further has to new of attest significant and regulation skin inflammation a during the the inflamed identify of in results from deletion These player by nodes. trafficking lymph BMDCs delays draining in CX3CR1 axis targeted that CX3CL1 receptor show the lymphatic we nodes of findings, lymph these disruption inhibit of draining corroboration to further CX3CL1 DCs In cutaneous against of trafficking of antibodies migration endothelium promotes neutralising lymphatic CX3CL1 activated across secreted Moreover, MDDC mice. that hypersensitised demonstrate contact we from sections intact skin in TNF- and CX3CL1 of of vessels induction lymphatic confirm we Additionally, LECs. synthesised is novo family, de chemokine CX3C transmembrane atypical ubr of numbers aucitwsta ohclue Esadlymphatic and sheddase ADAM-like an LECs of action the cultured through CX3CL1, of both form that endothelium was chemotaxis. as manuscript CX3CL1 much as of adhesion function cell of the one hence as regarded and been that al., has cells form et most membrane-anchored Yoshikawa in the 1997; is predominates al., it et however, Imai Notably, 2000; integrin-mediated 2004). of al., mechanism et conventional (Goda a adhesion by cells NK and ecmae h ieiso aoaea-olmnlmgainfor migration DCs basolateral-to-luminal receptor-null of and kinetics wild-type the compared we BMDCs lymphatics. afferent the the exited through having likely cells most of tissue, that majority the dermis, suggesting rarely the were BMDCs within hours, hours, observed 24 48 By adhesion. of endothelial at than rather surface areas endothelium basolateral towards migration these DC the influences CX3CL1–CX3CR1 in at accumulate vessels to lymphatic seen were BMDCs al iepit 2hus.Sgiiaty e fany if few Significantly, hours). (2 points time early XC1i atclryuuulisfra tcnocrbt as both occur can it as insofar unusual particularly is CX3CL1 nitiun idn rmtesuisi hspresent this in studies the from finding intriguing An ovrf httedlyi ypai rfikn of trafficking lymphatic in delay the that verify To XC1dpnetlmhtctafcig5265 trafficking lymphatic CX3CL1-dependent n hdi oul omb nlmdhmnadmurine and human inflamed by form soluble in shed and nvivo in Cx3cr1 nvivo in elcsa mareti rnedteilmigration, transendothelial in impairment an reflects 2 / 2 eeaeams xlsvl h soluble the exclusively almost generate n idtp MC ihntedri at dermis the within BMDCs wild-type and a tetdmueadhmndermis, human and mouse -treated nvitro in sn TNF- using + ooye,Tcells T monocytes, nvitro in Cx3cr1 a Cx3cr1 -activated n that and nvivo in 2 2 / / 2 2 . Journal of Cell Science hsidctn pcfct fmb4bnig l aarpeettemean the represent data All binding. mAb24 of specificity indicating thus vd rtoyi laaeb osiuieclathrin-mediated can constitutive CX3CL1 a types, by cell cleavage other proteolytic in form. that evade membrane-anchored indicate the of reports any, Previous if little, retain and activity, of incubate Activation HDLECs, * (J). MDDCs; resting inhibitors transmigrating (I) metalloproteinase mature MDDCs and labelled TNF- ADAM fluorescently and or of (D) (E,F,K) Number CX3CL1 controls (G). human CD83 recombinant of with expression or by alone assessed of was inclusion maturation (with and blotting HDLECs (C) western activated detected by and assessed resting as across transmigration MDDCs DC matured promotes CX3CL1 7. Fig. 5266 ora fCl cec 2 (22) 126 Science Cell of Journal P , .3 ** 0.03, P , .1.MueIG a nldda nioyemthdcnrl DAceae iaetctosadihbt nernactivation, integrin inhibits and cations divalent chelates EDTA control. isotype-matched an as included was IgG1 Mouse 0.015. a siuae DEsicbtdete ihceoienurlsn nioiso reeatIgG irrelevant or antibodies chemokine-neutralising with either incubated HDLECs -stimulated b atna odn oto)()adfo yoer B.Epeso fCR a also was CCR7 of Expression (B). cytometry flow and (A) control) loading as -actin 6 ..( s.e. b nernwsasse hog idn fmb4t maue()and (H) immature to mAb24 of binding through assessed was integrin 2 n 5 lentvl htte xii rae ee fCX3CL1 of level greater a or exhibit It LECs, in 2005). they efficiently al., less et that operates Liu pathway alternatively this 2009; that al., be et might (Huang pathway endocytosis )fo n ersnaieeprmn htwsrpae w ofv times. five to two repeated was that experiment representative one from 4) nvitro in . eeso XC1epeso nimtr n LPS- and immature in expression CX3CR1 of Levels either d Journal of Cell Science otu lt (B). plots contour XC1o oto abtIG aarpeettema recoveries mean the represent Data IgG. against ( rabbit antibodies oxazolone- neutralising control of of or injection painting CX3CL1 prior skin received FITC that after mice hours sensitised 24 nodes, lymph draining ntebsso t ihyplrsdscein epooethat propose we secretion, polarised zone. subendothelial highly the in its events 2011). influences of portals’, al., preferentially et CX3CL1 ‘entry basis (Tal at lymphatic DCs the IV of of transmigration On collagen and membrane with docking complexed the basement facilitating is secreted the for it basolaterally shown on where contrast, been vessels, sequestered has By as is 2001). node, al., CCL21 hence lymph et and the (Palframan flow in lymph CCL2 downstream afferent luminally role conveyed by of a be lymphatics could play fate initial it the although The from unclear, ( 2001). present away at mainly al., is CCL21 et biased secreted (Kriehuber is being secretion entry, the as luminal from well-documented lymphatic predominantly is for not secreted which data essential are CCL21, (and Even which 1999) al., surface. of et luminal all Saeki or 2001; Kabashima most al., 2010; et Jackson, shown), Kriehuber al., and 2007; et Johnson al., (Gunn 2006; et neutrophils al., and et cells Johnson T 1999; monocytes, (MIP-3 DCs, of CCL20 trafficking by (SDF-1), (RANTES), synthesised CXCL12 CCL5 (GRO CCL21, chemokines including (MCP-1), CXC LECs, and CCL2 inflamed CC lymphatic and of resting within repertoire of unique virtually large CX3CL1 surface the makes p This basolateral DCs. selectively of transmigration it the where from endothelium, exclusively lymphatics. almost in 2006). precollector heterogeneity al., et and similar (Johnson observed initial VCAM-1 of previously both expression have of we derive mixture thus Indeed, isolated and subtle a LYVE-1 are with the cells immunoselection from TNF by these reflects skin because the this within subtype, from that HDLEC expression in suspect variability CX3CL1 and adhesion HDLECs, We of favoured. stimulated an be the heterogeneity might as endothelium, molecule vascular the noted CX3CL1 blood of form the for chemokine to there soluble requirement contrast capillaries, in lesser blood and from the receptor, a exit combat of be their to migration have restricts might not that interstitial does flow the vessels shear lymphatic as into Moreover, leukocytes activity. sheddase CX3CL1. upon dependent is 8. Fig. n 5 nte atclryipratfnigwsta XC1i secreted is CX3CL1 that was finding important particularly Another )fo n xeieto w A,wt ersnaiefo cytometry flow representative with (A), two of experiment one from 5) b nvivo In ,CC5(N7)adCC8(L8 htinfluence that (IL-8) CXCL8 and (ENA78) CXCL5 ), rfikn fctnosDstruhafrn lymphatics afferent through DCs cutaneous of trafficking eoeyo FITC of Recovery ooe basolateral-to-luminal romotes + CD11c , + knDsi the in DCs skin 0)towards 70%) a ,CXCL2 ), 6 s.e. a - isi okn n rnmgain(a ta. 01.Moreover, 2011). we al., inflammation, to et response in (Tal strictly induced transmigration is and CX3CL1 CCL21 because immobilised docking which in at stage aids the the towards before DCs chemotaxis, capillaries, to fluid-phase guidance lymphatic not Thus, in initial provide role 2001). is and a haptotaxis, al., play than consequently rather et to expected and (Patel matrix-sequestered be matrix could CCL5 other CX3CL1 extracellular and DC tissue in in CCL21 found sequestered as a is inhibit contain such as not chemokines effects tract does antibodies additive CX3CL1 GAG-binding yield together. basic to combined neutralising fail when but blockade on singly, both CCL21 added that when finding transmigration our and is with ‘lymphatic’ CCL21– consistent stages CX3CL1–CX3CR1 CX3CL1 cascade, of of sequential at migratory absence act hierarchy the why CCL21 the and during in CX3CL1 the that trafficking explain propose We DC in CCR7. maintain might position to rate- insufficient the dominant and over a control chemokines Such in CCL21–CCR7 2011). the places axis al., therefore transmigration notably et DC (Johnson in Tal step conditions most limiting 2010; inflammatory but and Jackson, resting and movement both functional under amoeboid crawling the by each by in migration DC of interstitial transendothelial positions only action of not relative promoting of actions, chemotaxis their several sites has as the CCL21 hierarchy. well 2007). in al., as differences et chemokine, reflects (Kabashima axis probably CXCL12–CXCR4 This the of inhibition C a eosrtdb esrn FITC measuring by demonstrated lymphatic was the DCs of surface basolateral guidance. the CCL21 before towards vessel, DCs gradients chemokine attract local the that establish retarded, might is CX3CL1 flow of lymph accumulation when inflammation, during example, For riiglmhnds2 or fe ICsi painting. skin FITC FITC of lymph after trafficking suppressed antibodies hours node 24 neutralising by blockade nodes CX3CL1 lymph draining irto fDsta a enrpre fe irpino h key the node of disruption lymph after and reported CCR7 entry been in axis has lymphatic CCL21–CCR7 that in DCs of reduction migration sustained the with apoptosis. almost DC in were increase hours an cannot 48 CX3CR1–CX3CL1 by and disrupting explained of be hours effects 24 the after Hence, nodes identical. lymph cervical the oa ubr fwl-yeand wild-type of here. numbers fluorescently done using total have we trafficking migration as of DC DCs, kinetics of labelled the measurements quantifying indirect than made rather study previous the of fti niiinmgtas expl also might inhibition this of skin to , transit de their of delaying inflamed mice, the deletion from genetic trafficking that newly than experiments DCs dermal be DCs. to monocyte-derived likely differentiated more and trafficking is recruited population of nodes major lymph number the the hours inflammation, the to 24 of However, reducing onset dermis. the neutralising thus after the experiments, CX3CL1 entering from DCs these monocytes that monocyte-derived of in possible recruitment blood blocked also the entry have is DC might impairing it antibodies to addition lymphatics, In to controls. IgG with compared Ctafcigwsoeloe uigteoiia hrceiainof characterisation original the global during overlooked was trafficking DC 4huscmae ihwl-yece wild-type with compared hours 24 oefrC3L nlmhtctafcigo endogenous of trafficking lymphatic in CX3CL1 for role A h eae rfikn fC3R-eiin C locontrasts also DCs CX3CR1-deficient of trafficking delayed The otsgiiaty eosre noradoptive-transfer our in observed we significantly, Most XC1dpnetlmhtctafcig5267 trafficking lymphatic CX3CL1-dependent Cx3cr1 2 / 2 ncotmc Jn ta. 00.As,teauthors the Also, 2000). al., et (Jung mice knockout 2 b + / 2 /CD11c nernatvto n intraluminal and activation integrin 2 i o h nlec fC3L on CX3CL1 of influence the how ain Cx3cr1 ie(h ta. 04 rfollowing or 2004) al., et (Ohl mice mso xzln hypersensitised oxazolone of rmis dann evcllmhndsby nodes lymph cervical -draining motnl,i u experiments, our in Importantly, l.Ide,tetasetnature transient the Indeed, lls. + 2 / Cx3cl1 2 el by cells MC eoee from recovered BMDCs + /CD11c nDsimpeded DCs in . 0 when 70% + el in cells Journal of Cell Science l tde sn ua isewr prvdb h xodRgoa Ethics Regional Oxford Office guidelines. the Home institutional UK by established appropriate to with approved according performed were were licences studies tissue animal All human Committee. using studies All studies animal and Human Methods and Materials lymphatic is the CX3CL1 by and response pleiotropic inflammation. passive, wider to from a endothelium far of part is clear as now process induced is complex It this vessels. from role lymphatic that DCs afferent novel of through trafficking a skin regulating inflamed and in chemokine endothelium atypical this lymphatic for inflamed in secretion only. CCL21–CCR7, conditions unlike inflammatory under axis, trafficking DC CX3CL1–CX3CR1 guides the that conclude 5268 vrl,orfnig eosrt nuto fCX3CL1 of induction demonstrate findings our Overall, ora fCl cec 2 (22) 126 Science Cell of Journal he nihmnvnWlern atr(W) Srtc n a nimueEpCAM anti-mouse rat (BD and (Serotec) VCAM-1 (vWf), factor anti-human Willebrand mouse von Bank), anti-human Hybridoma Studies (Chemicon), sheep (Developmental City, E-selectin ICAM-1 Iowa anti-human mouse Iowa, anti-human Pharmingen), of University mouse Bank, Hybridoma IA), podoplanin, Studies anti-mouse (Developmental rabbit 8.1.1 International), clone anti- Industries rabbit (Fitzgerald Biotechnology), (AMS podoplanin AMS anti-CX3CR1 human from rat anti- Systems), mouse were were (R&D used CX3CL1 antibodies antibodies human Other neutralising respectively. Systems, anti-CCL21 R&D and goat Biotechnology and anti-CX3CL1 Rabbit Antibodies 25 domain), (chemokine ng/ml; 20 Abortus GM-CSF, CX3CL1 mouse Salmonella ng/ml; human 20 IL-4, mouse and ng/ml; 50 GM-CSF, human ml; the TNF at human used ng/ml; and IL-1 100 Systems R&D concentrations: from were following proteins mouse and human factors Recombinant growth and chemokines Cytokines, a rmSigma-Aldrich. from was a gm;mueTNF mouse ng/ml; 2 , a n IL-1 and mgn fwl-ye(elTakrRed and Tracker labelled) (Cell wild-type of imaging n ersnaieeprmn fthree. of experiment representative one means the Data represent inserts. FluoroBlok of the underside on plated monolayers MDLEC TNF- across transmigrating eyfew very of a one hours indicates 48 Arrow and co-injection. 24 after 2, at remaining BMDCs neto.Foe etoso yp nodes after lymph of hours sections 48 Frozen and injection. 24 at nodes draining lymph in BMDCs labelled) Green on knscin ihLYVE-1 with sections Whole- skin (C) mount nodes. lymph of in paracortex BMDCs indicate subcapsular arrowheads the sinus; in BMDCs of indicate examples Arrows yellow. TOPRO-3, with counterstained were HEVs, Nuclei and respectively. sinus subcapsular lymphatic or label vessels to (blue) MECA79 (yellow) and anti-LYVE-1 with stained were fwl-ye(-rce55lble)and Cx3cr1 labelled) (Q-tracker525 wild-type of lymphatics. skin inflamed through trafficking for DC required efficient is CX3CR1 9. Fig. mgsfo i ieaeson Scale shown. 50 are bars: mice six from Representative images hours. 48 at remained and Cx3cr1 (red) wild-type of numbers comparing (blue) vessels lymphatic n ersnaieeprmn fthree; * of experiment representative one means the represent Data topical mice. of C57Bl/6 skin oxazolone-sensitised a the of into co-injection mixture after 50:50 hours 48 or nodes 24 lymph draining in arriving BMDCs aeldwl-yeand wild-type labelled P , .0,** 0.001, b gm;I-,2 gm;IFN- ng/ml; 20 IL-6, ng/ml; 1 , 2 2 m / / a 2 2 .()Nme ffluorescently of Number (D) m. 0 gm;hmnI-,1 ng/ 10 IL-4, human ng/ml; 100 , gen dpieytransferred adoptively (green) Cx3cr1 Qtakr5 labelled) (Q-tracker655 m Cx3cr1 P /l P (1 LPS g/ml. , .09 B Fluorescent (B) 0.0079. 2 / 6 6 2 2 / 2 MC that BMDCs Cx3cr1 ..( s.e. ..( s.e. Cl Tracker (Cell a A Number (A) n n m -stimulated 5 5 /l from g/ml) 2 )from 4) )from 3) / 2 + DCs c , Journal of Cell Science muoeeto ihLV- A n ASbas(itniBoe) and Biotec), (Miltenyi beads MACS and mAb LYVE-1 by with samples skin resected freshly immunoselection from prepared were MDLECs and HDLECs Primary 2001). al., et Prevo Cells 2006; Systems. were al., R&D antibodies et or anti-LYVE-1 Sigma-Aldrich Johnson from 1999; All were al., Pharmingen. antibodies Isotype-matched et BD (Banerji from CD11c- previously anti-mouse CD86-PE, were generated goat anti-mouse CCR7-PE conjugated and and Directly CCL5 Systems. APC R&D anti-mouse from goat were CCL2, CCL20 anti-mouse Goat (eBioscience). rmrpi XC1w2(-TGTCAACCGCT3 and the the (5-ATGGCTCCGATATCTCTGTCGT-3) following using amplified Biolabs), CX3CL1Fwd2 and were England (5-AAAAGCTCCGTGCCCACA-3), pair transcripts CX3CL1Rev2 (New CX3CL1 transcriptase primer instructions. reverse TNF- manufacturer’s for ng/ml AMV 2 cultured with supplemented HDLECs using or from alone either Qiagen) medium, a MV (RNeasy, EGM-2 isolated in hours was 24 RNA cellular Total RT-PCR to anti- transferred 0.2 mouse was antibodies, MES with Protein incubated with anti-CX3CR1 and (Invitrogen). Invitrogen) rabbit (Millipore) standards (NuPAGE; membranes SeeBlue gels on Immobilon resolved SDS alongside and (Invitrogen) polyacrylamide buffer, buffer sample 4–12% LDS NuPAGE Bis-Tris inhibitor in lysed protease were MDDCs and Tris-HCl, EDTA mM blotting mM [50 Western 1 ELISA. buffer the (v/v), to lysis NP-40 applied in then 1% and lysed NaCl, the (Roche)] mM cocktail were to 100 cut- the kDa cells 7.4, directly 5 with pH Adherent tubes, centrifugation applied accordance ml (Millipore). 0.5 in either Ultra commercial off Amicon Systems), using were using concentrated R&D or CCL5 Supernatants ELISA (DuoSet; and instructions. kits CCL2 manufacturer’s ELISA CX3CL1, (0.4 inserts capture for Transwell antigen HDLECs assayed on primary or were confluent dishes of Biosciences), six-well wells in triplicate cultured from (either lysates cell and Supernatants ELISA Chemokine 100 magnification: 10 Plan-Apochromat 630 magnification: (total a either M27 using microscopes, DIC confocal LSM780 Zeiss 2000 Radiance or Bio-Rad under viewing in Probes, and mounting Laboratories) (Molecular PFA-PBS, (Vector in stain Vectashield-DAPI fixing nuclei before detection secondary DAPI for or used TOPRO-3 were Invitrogen) either Alexa 568, and Fluor conjugates Alexa 488, 647 Fluor Fluor Alexa temperature. 0.2% room permeabilisation in at with cell minutes prior PBS without 45 or for in with saponin antibodies BSA, (w/v) primary 1% appropriate PBS, and FCS the in (v/v) 10% w/v with incubated (1% paraformaldehyde PFA-PBS); in 7.4; fixed pH were tissues MDLECs and and HDLECs cells Cultured of staining antibody Immunofluorescence 2.5 at used and Calbiochem from purchased was (GM6001) Ilomastat Inhibitors the following marrows bone (Invitrogen), and fibula C57Bl/6 euthanised and Green of tibia Tracker from extracted were Cell BMDCs protocol. manufacturer’s using MDDCs labelled 2006). al., fluorescently et from (Johnson were described obtained previously were as generated MDDCs and 2006). donors al., healthy et (Johnson described previously as cultured ASbas(itniBoe)adte ihrlble ihQtakr5 rQ- or Q-tracker655 protocol. manufacturer’s with the labelled following either Tracker (Invitrogen), Cell Red or then Green Tracker Cell and with or Invitrogen), Biotec) Probes, (Molecular by tracker525 expression (Miltenyi II class beads MHC with supplemented for and MACS selected FCS were cells 10% Non-adherent with IL-4. RPMI and in GM-CSF days 7 for cultured and Biosciences) olwdb otat-os Ry70adga nirbi IRDye800 anti-rabbit Li-Cor the goat using visualised and were IRDye700 Blots system. buffer. imaging Odyssey anti-mouse blocking Biosciences Odyssey goat in (Odyssey) by followed BSA. (w/v) 1% and FCS TOPRO-3. (v/v) with counterstained for were 10% used nuclei was and Invitrogen) with detection Probes, non- secondary (Molecular mouse PBS Blue observe against Pacific to in antibodies to conjugated Streptavidin rat MECA79 alone, 647 with Fluor biotinylated Alexa incubated PBS with conjugated directly and then 2007)] al., in acetone et (Johnson washing C1/8 ice-cold [clone LYVE-1 in or fixed minutes cryostat, by 5 PBS prepared were for node in lymph of with sections X-100 Frozen tissue permeabilising tissue. permeabilised Triton either 2006), (v/v) al., et 0.1% (Johnson previously described as out at used and MMP9 10 Calbiochem and at from used MMP2 purchased and were Germany). I University, inhibitor 1 MMP8 Aachen and gift Ludwig, II kind inhibitor a Andreas were GI254023X Dr and from GW280264X inhibitors ADAM17 and ADAM10 is-tadcN ytei a are u yOiod rmn (Invitrogen) priming dT Oligo by out carried was synthesis cDNA First-strand . m .AtnmcnDadccoeiiewr ucae rmSigma-Aldrich from purchased were cycloheximide and D Actinomycin M. m /lad20 and g/ml 6 eouin 0.24 resolution: , Cx3cr1 m /l respectively. g/ml, 2 / 2 6 m ie asdtruha70 a through passed mice, ) hl-on isesann a carried was staining tissue Whole-mount m). rPa-pcrmt63 Plan-Apochromat or ) m /l(M itcnlg)overnight, Biotechnology) (AMS g/ml b atnwt h primer the with -actin m elsrie (BD strainer cell m m 6 oesz;BD size; pore m 14ol(total oil /1.4 b atnand -actin 6 m /0.3 M. rsBrt-DAgl,aogie10b N adr NwEgadBiolabs). England (New (5- ladders DNA bp ActinRev 100 agarose- alongside 1.8% gels, and Tris-Borate-EDTA on resolved were (5-AGGCATCCTCACCCTGAAGTAC-3) Products TTGCCAATGGTGATGACCTGGC-3). ActinFwd pair h diino DC n anandtruhu h oreo h experiment. the of course the 0.5 throughout CX3CL1 well, maintained each anti-human and To MDDCs Rabbit of addition MDDCs. the of (50 antibodies addition Where neutralising (50 before 2010). antibodies hours Jackson, neutralising 2 and added Johnson TNF- 2006; was with stimulated al., were cells et (3 indicated, as (Johnson performed inserts were previously assays culture transmigration detailed and cell confluent until otherwise FluoroBlok cultured (unless Biosciences), underside gelatin-coated the of onto seeded stated) were MDLECs and HDLECs Primary vitro In 4 at minutes 10% 30 with for PBS antibodies primary in appropriate washed with PFA-PBS, incubated then in FCS, fixed (v/v) were HDLECs and BMDCs MDDCs, cytometry Flow aucit ...dsge eerhadwoetemanuscript. the wrote and the research wrote and designed data D.G.J. analysed manuscript; research, performed and designed L.A.J. contributions Author Graham and imaging inhibitors LSM780. on ADAM17 assistance Zeiss technical the and excellent the with for ADAM10 UK) for Zeiss Aachen) the (Carl of Brown of (University gift Ludwig kind Andreas thank authors The point. Acknowledgements time hour 8 the study. from this data throughout on sets performed data were compare to used P was U-test Mann–Whitney The analyses Statistical littermates from 10 and BMDCs 10 oxazolone adoptive Invitrogen)-labelled 0.8% of and For Probes, application days topical (Molecular DAKO). 5 by were and challenged Q-tracker655 (CyAn, oxazolone, were nodes to ears cytometry sensitised 70 both lymph were later, mice flow a cervical male through C57Bl/6 by experiments, draining passed and transfer analysed hours The disrupted, and 24 was hours. after w/v Biosciences) 24 water) tissue 0.8% in further and (v/v with ethanol removed a challenged 95% after then in FITC Systems) (AMS sacrificed mg/ml antibodies (R&D 1.5 neutralising and IgG anti-CX3CL1 FITC oxazolone rabbit rabbit For either or 2006). intraperitoneally of Biotechnology) mg were al., 0.4 mice et with BALB/c (Johnson injected oxazolone-sensitised described experiments, previously painting phenyl-2-oxazoline-5- as (4-ethoxymethylene-2 challenged Sigma-Aldrich) and oxazolone sensitised one; of were weeks application 8–10 topical aged by mice C57Bl/6 and BALB/c Male vivo In notelwrcabrwr eoddo loecn lt edr(yeg HT; (Synergy reader plate fluorescent a on monolayer recorded and filter were the chamber through lower transmigrating the MDDCs into of numbers and assay the i-e)a 37 at Bio-Tek) airtdaantasadr uv,adtasirto a xrse sthe as expressed was Cx3cr1 transmigration chamber. lower and the in curve, MDDCs standard of number a against calibrated el n loecneeiso a airtdaantsadr uvsfrwild- for curves standard against calibrated parallel and was in type emission monolayers MDLEC fluorescence to and applied wells were above) (described donors matched ln rwt ua TNF- human medium EGM-2MV either with in of hours or expression 24 surface for alone of cultured analysis were For cells 488. HDLECs, antibodies Fluor in goat CX3CL1 Alexa with to incubation conjugated by followed IgG FCS, rat (v/v) against 5% with PBS in washed then nml eesciie n yp oe rcse sdtie bv.For above. 10 and detailed numbers equal in as mixed (Invitrogen), Tracker processed period, dyes Cell Green hour either Tracker nodes with 48 Cell pre-labelled or or lymph Red were 24 BMDCs and post-injection, further DCs a sacrificed visualising After were injected. dermally animals were above) (described n rznscin rprdo yp nodes. lymph of prepared sections frozen and eeijce.Dri a hnsandi hl-on o LYVE-1 for whole-mount in stained then was Dermis injected. were nlsdb lwctmty(yn aoCtmto)uigFo osoftware. were Jo Cells Flow 2010). using Cytomation) Jackson, Dako and (CyAn, (Johnson cytometry previously flow by described analysed MDDC as of out Measurement carried DCs. for fixed was were as Cells stained CX3CL1. and of PFA-PBS shedding in subsequent prevent to Ilomastat, of presence , ocmaemgaino MCfo idtp iewt hs from those with mice wild-type from BMDC of migration compare To .5wscniee infcn.Frtasirto sas ttsia analyses statistical assays, transmigration For significant. considered was 0.05 XC1dpnetlmhtctafcig5269 trafficking lymphatic CX3CL1-dependent 2 6 sasfrC3L-eitdD rfikn nifae os skin mouse inflamed in trafficking DC CX3CL1-mediated for assays ypai nohla rnmgainassays transmigration endothelial lymphatic / 2 Cx3cr1 -rce55lble MC rmage-matched from BMDCs Q-tracker525-labelled ie elTakrGenlble H ls II class MHC Cell-Tracker-Green-labelled mice, ˚ 2 sn C otae(itc) loecneeiso was emission Fluorescence (Biotech). software KC4 using C 6 / 2 10 cells. 6 loecnl aeldMDswr ple ttesatof start the at applied were MDDCs labelled fluorescently m a m /l & ytm)wr ple 0mntsbefore minutes 30 applied were Systems) R&D g/ml, /l M itcnlg)adga anti-CCL21 goat and Biotechnology) AMS g/ml, n/rIoatt hnlfe ihActs nthe in Accutase with lifted then Ilomastat, and/or a o 4husbfr s n CX3CL1 and use before hours 24 for m b elsrie (BD strainer cell m nernactivation integrin 2 m + oesz;BD size; pore m Cx3cr1 C rmage- from DCs + lymphatics 2 / 2 5 mice cells ˚ C 6 Journal of Cell Science udasn . izea . ekot .A,Boda,N,Sfi,P,Ris K., Reiss, P., Saftig, N., Broadway, A., T. Berkhout, Robinson, D., and Misztela, H. C., Yan, Hundhausen, D., Chaukos, R., M. Crow, Y., G. Liu, P., Su, W., Y. Huang, F. I. Charo, and D. M. Cleary, A., C. Haskell, and T. L. Williams, A., Matsuzawa, T., Kakiuchi, C., Tam, S., Kyuwa, D., M. Gunn, Imai, T., Okazaki, Y., Nagano, H., Inoue, O., Yoneda, O., Yoshie, T., Imai, S., Goda, esmn,F,Mn,M . ug . iwk,M . ea,M n e,K. Ley, and M. Merad, H., M. Sieweke, S., Jung, G., M. Manz, F., Geissmann, atn .J,Guh .J n ans .W. E. Raines, and J. P. Gough, J., Dempsey, K. R., Garton, D. Greaves, G., Murphy, P., C. Blobel, J., P. Gough, J., K. Garton, Fo og .M,Rbno,L . tee,D . edr .F,Ysi,O,Ia,T and T. Imai, O., Yoshie, F., T. Tedder, A., D. Steeber, A., L. Robinson, M., A. Fong, Caban I., Dransfield, reue,E,BetndrGlf,S,Gogr . oemn . Schoppmann, A., Soleiman, M., Groeger, S., M., Breiteneder-Geleff, Kobayashi, E., Kriehuber, A., Onoue, T., Mori, K., Sugita, N., Shiraishi, Sher, K., W., Kabashima, G. Kreutzberg, J., M. Sunshine, P., Graemmel, J., Aliberti, S., Jung, or-oti,K,Bse,L,Bnir . ’egul’,A,Smo,M n The and M. Samson, A., L’helgoualc’h, D., Bonnier, L., Basset, K., Bourd-Boittin, ono,L . rv,R,Capr .adJcsn .G. D. Jackson, and S. Clasper, R., G. Prevo, D. Jackson, A., and L. D. Baban, Johnson, F., P. Lalor, P., A. Holt, S., Clasper, A., L. Johnson, G. D. Jackson, and A. L. Johnson, Kakizaki, M., Nishimura, M., Nagira, M., Baba, C., Haskell, K., Hieshima, T., Imai, aa,J . ao,K . admn . ag . o,K,Rsi . Greaves, D., Rossi, K., Soo, and W., M. Wang, G., Jones, Hardiman, R., B., K. Tammi, Bacon, J., F., Su, J. Bazan, S., Clasper, S.-X., Wang, J., Ni, S., Banerji, References at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.135343/-/DC1 online to available material Funding Supplementary Unit release. and immediate L.A.J. for to PMC Council, in Award Research Deposited Medical Investigator D.G.J. UK New the a by through supported was research This Funding 5270 rtr . cue,A,Beted . rme,E,Renner-Mu E., Kremmer, D., Breitfeld, A., Schubel, R., ¨rster, atan . arnoz . otn,R,Mthw,V tal. of et cleavage V. adhesion. constitutive cell-cell CX3CL1-mediated Matthews, in regulates and involved R., (fractalkine) is CX3CL1 Postina, ADAM10 F., metalloproteinase disintegrin-like Fahrenholz, D., Hartmann, metalloproteases. surface cell by degradation A. of L. arrest flow Rapid activation. transduction. G-protein of 10053-10058. signal independent localization. is and cells cell CX3CR1-expressing adhesion dendritic cell and fractalkine-mediated homing lymphocyte Med. in Exp. - defects J. and have integrin-dependent chemokine through H. cells Nakano, endothelial to cells mechanisms. THP-1 independent al. et of N. Domae, adhesion T., E. Bloom, H., 656-661. 21) eeomn fmncts arpae,addnrtccells. dendritic and macrophages, monocytes, of Development (2010). hdigi h euaino nlmaoyresponses. inflammatory of regulation the in shedding (CX3CL1). fractalkine of shedding and cleavage Chem. W. the E. mediates Raines, (ADAM17) and J. P. ucinlmconiomnsi eodr ypodorgans. lymphoid secondary in microenvironments functional M. Lipp, and E. flow. physiologic under activation 188 and adhesion, firm capture, D. leukocyte D. function. Patel, for sufficient not but necessary is 1527-1535. ligand with integrins .F,Sig,G,Krack,D n arr D. Maurer, and D. Kerjaschki, G., Stingl, F., S. cells. al. dendritic et cutaneous N. of 1249-1257. migration Fujii, for H., required is Tamamura, engagement R., Yoshiki, J., Sakabe, insertion. gene reporter protein fluorescent green Biol. and deletion targeted R. by D. Littman, and A. ocrncifamto nteliver. the in inflammation chronic to N. nue paeaddgaaino h ypai nohla ylrnnreceptor hyaluronan endothelial lymphatic the of degradation LYVE-1. and uptake across induced transmigration leukocyte cell for endothelium. dendritic mechanism vessel lymphatic integrin-mediated inflammation-induced An of (2006). regulator key a is transmigration. endothelium lymphatic both al. mediates in et which J. CX3CR1, adhesion. T. receptor and Schall, migration fractalkine leukocyte H., of characterization Nomiyama, molecular S., Takagi, M., 1186-1195. .R,Zonk .adShl,T J. T. Schall, motif. CX3C and a A. with chemokine Zlotnik, R., D. hyaluronan. for receptor lymph-specific G. D. Jackson, 20) XC1fatliesedn yhmnhptcselt el:contribution cells: stellate hepatic human by shedding CX3CL1/fractalkine (2009). 1413-1419. , 20 276 20) osiuieedctsso h hmkn XC1peet its prevents CX3CL1 chemokine the of endocytosis Constitutive (2009). 4106-4114. , .Bo.Chem. Biol. J. ora fCl cec 2 (22) 126 Science Cell of Journal 37993-38001. , 189 19) ielcigepeso fscnaylmhi organ lymphoid secondary of expression lacking Mice (1999). 19) rcakn n XC1mdaeanvlmcaimof mechanism novel a mediate CX3CR1 and Fractalkine (1998). n.Immunol. Int. 19) C7codntstepiayimn epneb establishing by response immune primary the coordinates CCR7 (1999). 19) YE1 e ooou fteC4 lcpoen sa is glycoprotein, CD44 the of homologue new a LYVE-1, (1999). 451-460. , a,C,Bret .adHg,N. Hogg, and J. Barrett, C., ˜as, 20) nlsso rcakn eetrC()R function CX(3)CR1 receptor fractalkine of Analysis (2000). 282 .Immunol. J. 20) uo erssfco-lh-ovrigenzyme factor-alpha-converting necrosis Tumor (2001). 33671-33680. , 22 .Ep Med. Exp. J. 839-849. , Nature 21) nlmainidcdsceino CCL21 of secretion Inflammation-induced (2010). Cell .Cl.Ml Med. Mol. Cell. J. 20) XCceoie fractalkine-enhanced CX3C-chemokine, (2000). 164 385 91 .Cl Biol. Cell J. 19) e ls fmembrane-bound of class new A (1997). 521-530. , 4313-4320. , 640-644. , 203 .Bo.Chem. Biol. J. 20) mrigrol Emerging (2006). 2763-2777. , 19) oeua nopigof uncoupling Molecular (1999). .Luo.Biol. Leukoc. J. 19) neato fleukocyte of Interaction (1992). 144 38A 13 789-801. , 19) dniiainand Identification (1997). 20) CXCL12-CXCR4 (2007). Cell 20) slto and Isolation (2001). 284 1526-1535. , 20) Inflammation- (2007). 99 29644-29653. , .Bo.Chem. Biol. J. m .Pathol. J. Am. .Cl Biol. Cell J. ,23-33. 79 ectodomain for es le,I,Wolf, I., ¨ller, 1105-1116. , Science .Ep Med. Exp. J. 20) The (2003). Blood o.Cell. Mol. .Biol. J. ´ret, 274 327 116 171 102 , , , , , h,L,Mhut . zlt,N,Hnzn . ifr,Z,Zinr J., Zwirner, Z., Kiafard, G., Hintzen, N., Czeloth, M., Mohaupt, L., Ohl, uhhee,A,Suemn,L . u . udk-eknap . air R., Xavier, K., Luedtke-Heckenkamp, X., Gu, J., L. Saubermann, A., Muehlhoefer, H., D. McDermott, P., Seknadji, M.-W., Amara, F., Fumeron, S., Faure, D., Moatti, Martı i,G . uaigm . lxne,R . ort . og .M,Ptl .D. D. Patel, M., A. Fong, N., Touret, T., R. Alexander, V., Kulasingam, Y., G. Liu, ohkw,M,Nkjm,T,Mtuoo . kd,N,Tuiae . ia M., Iida, T., Tsukidate, N., Okada, K., Matsumoto, T., Nakajima, M., Yoshikawa, cence,C,MHg,R,Seah .M n emi,R N. R. Germain, and M. E. Shevach, R., McHugh, C., Scheinecker, La of,B,Brs .R,Mdltn .adRt A. Rot, and J. Middleton, R., A. Burns, B., Wolff, ee,M,Hucid . cwr,J,Muso,C,d re,I,Lge,D F., D. Legler, I., Vries, de C., Moussion, J., Schwarz, R., Hauschild, M., Weber, T. S. Hwang, and W. J. F. M. Brown, Luscinskas, M., and A. Moore, G. H., Garcia-Cardena, Saeki, L., Yang, M., R. Rao, Llodra V., Angeli, F., Tacke, W., G. D. E. Jackson, Edwards, and C., S. Qu, Clasper, P., J. D. Krangel, Ferguson, and S., O. Banerji, Yoshie, R., P., Prevo, L. Whichard, T., Imai, W., Koopmann, D., D. Patel, ht,G .adGevs .R. D. Greaves, and E. G. White, a,Y,Lod . hu . oih . ed,J,Gnao .A,Vt,J,Gosselin, J., Vath, A., J. Gonzalo, J., Deeds, S., Dolich, H., Zhou, C., Lloyd, Y., Pan, Littman, M., Dorf, Y., Luo, W., Weninger, G., Cheng, S., Jung, T., R. Palframan, imn,D,Geee,M,Shi,S,Kimk . og . uwg .and S. Ludwig, C., Sorg, K., Klimmek, S., Schmid, M., Goebeler, D., Viemann, T., Okazaki, Y., Tanaka, Y., Minami, Y., Nagano, T., and Imai, V. S., Angeli, Goda, H., G., Umehara, L. Ng, Z., Shipony, I., Milo, I., Gurevich, Y., H. Lim, O., Tal, Garin, J., Llodra, R., Spanbroek, C., Jakubzick, J., T. Kaplan, D., Alvarez, F., C. Tacke, M. Nussenzweig, and D. Hawiger, M., R. Steinman, memn,T,Bdr .L,Mnly .J,Wrs . Wedlich-So T., Worbs, J., S. Monkley, L., B. Bader, T., ¨mmermann, lnesen . enn,G n Fo and G. Henning, T., Blankenstein, 3376. lmeg .S,Pdlk,D . aDrot .P n ence,H C. H. Reinecker, mucosa. for and intestinal chemoattractant small P. cell-derived the endothelial R. in and MacDermott, lymphocytes epithelial K., an intraepithelial D. is Fractalkine Podolsky, (2000). S., R. Blumberg, Debre oyopimi h rcakn eetrC3R sagntcrs atrfor factor risk genetic a as CX3CR1 receptor disease. fractalkine artery coronary the in Polymorphism priming. and traffic lymphocyte T on impact 198 node: lymph draining F. the Sallusto, and A. Lanzavecchia, n oisn .A. CX3CL1. L. Robinson, and euaeepeso ffatlie(XC1 sammrn-nhrdproadhesive membrane-anchored a fibroblasts. human al. as on peptide et chemotactic (CX3CL1) soluble and T. fractalkine protein Imai, of H., expression Moriyama, regulate S., Haruna, N., Otori, ai ekct irto yitgi-needn lwn n squeezing. and flowing integrin-independent by 453 migration Fo leukocyte M., Rapid Keller, K., Hirsch, fifamtr tmlto:hmnvnlredteilclssoeitrekn8in 8 interleukin store cells endothelial venular bodies. human Weibel-Palade stimulation: inflammatory of uhr .A,Blebc,T n it M. Sixt, and T. Bollenbach, A., S. Luther, cells node. to dendritic by lymph skin exclusively draining autoantigen the the tissue in natural from a of cells presentation dendritic Constitutive mature of pathway (CCR7) nodes. 7 emigration lymph receptor regional the chemokine CC in and participate (SLC) chemokine lymphoid-tissue secondary to al. cells et dendritic monocyte-derived N. of Rooijen, nodes. migration the van lymph in W., pathways Peters, chemokine other S., and N. Haque, A., Garin, Immunol. S. M. hrceiaino emllmhtcadbodedteilclsrva tbeand stable reveal cells endothelial lineages. blood cell and specialized lymphatic functionally dermal of characterization nippoi mediators. antiapoptotic rnpr n rsnaini E:armt oto ehns o monocyte for al. et mechanism B. Dussault, control J., Ma, remote M., tissues. a inflamed in HEV: nodes al. lymph in et to A. presentation recruitment Rot, H., and Zweerink, J., transport B. Rollins, R., D. conditions. steady-state and 279-288. inflammatory under migration cell dendritic iaedpnetgn xrsini N-lh-tmltdpiayhmnendothe- human primary cells. TNF-alpha-stimulated lial in expression gene kinase-dependent J. Roth, THP-1. line cell monocytic in molecule Biol. N. adhesion Domae, an as and predominantly T. E. Bloom, crawling. intralymphatic and endothelium 2141-2153. lymphatic on CCL21 plaques. G. Shakhar, atherosclerotic within accumulate to Invest. CX3CR1 Clin. al. and J. et CCR5, N. Rooijen, CCR2, van employ M., Mack, J., Liu, A., wall. vascular the to recruitment Res. leukocyte Circ. of mechanisms Endothelial-dependent endothelium. lymphatic in hyaluronan for receptor Chem. endocytic Biol. J. an is LYVE-1 Mouse inflammation. brain in upregulated yhpoatcceoiegradients. chemokine haptotactic by cells. nFneh,A,Sbsin,S,Ho S., Sebastiani, A., ´n-Fontecha, 615-621. , 51-55. , ,P,Amn,M . upy .M,d rs,D tal. et D. Prost, de M., P. Murphy, C., M. Aumont, P., ´, 79 nu e.Immunol. Rev. Annu. 20) hmknshv ies blte ofr oi hs gradients. phase solid form to abilities diverse have Chemokines (2001). 298-302. , Blood 99 20) rncitoa rfln fIK/FkpaB n 3 MAP p38 and B- IKK2/NF-kappa of profiling Transcriptional (2004). .Bo.Chem. Biol. J. 101 43-52. , 21) Cmblzto rmtesi eursdcigt immobilized to docking requires skin the from mobilization DC (2011). .Ep Med. Exp. J. 234-247. , 117 276 103 185-194. , 19420-19430. , 3365-3373. , .Immunol. J. .Ep Med. Exp. J. 20) eyln ftemmrn-nhrdchemokine, membrane-anchored the of Recycling (2005). Blood reislr hob ac Biol. Vasc. Thromb. Arterioscler. 280 rtr . rthe,D . Fa R., D. Critchley, R., ¨rster, 200 21 .Ep Med. Exp. J. 19858-19866. , 685-711. , 19) ertci,ammrn-nhrdchemokine membrane-anchored a Neurotactin, (1997). 21) rcakn:asrio’ ud:ceoie as chemokines guide: survivor’s a Fractalkine: (2012). 97 1231-1241. , 20) rcakn,aC3-hmkn,functions CX3C-chemokine, a Fractalkine, (2001). 1925-1928. , Nature 162 20) euaino edii elmgainto migration cell dendritic of Regulation (2003). 188 Science .Ep Med. Exp. J. 2472-2475. , pe,U . gcin,M,Lp,M., Lipp, M., Uguccioni, E., U. ¨pken, 1757-1762. , 196 rtr R. ¨rster, 387 21) nesiildnrtccl guidance cell dendritic Interstitial (2013). 339 20) ooyesbesdifferentially subsets Monocyte (2007). 1079-1090. , 611-617. , .Ep Med. Exp. J. 328-332. , 19) nohla el‘‘memory’’ cell Endothelial (1998). 20) nlmaoychemokine Inflammatory (2001). 194 20) C7gvrsskin governs CCR7 (2004). 20) oeoei dendritic Tolerogenic (2003). 797-808. , ,J,SnhzShiz G., Sanchez-Schmitz, J., ´, 20) N-lh n IL-4 and TNF-alpha (2004). 32 slr .e al. et R. ¨ssler, ESLett. FEBS .Immunol. J. 589-594. , 20) oeo CCR8 of Role (2004). 194 19) utn edge: Cutting (1999). 1361-1374. , .Ep Med. Exp. J. muo.Cell Immunol. 561 Immunity .Ep Med. Exp. J. lnr R., ¨ldner, 164 105-110. , 3368- , (2001). (2007). (2008). (2002). (2001). Nature Clin. 208 21 , ,