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The lymph vessel network in mouse skin visualised with antibodies againstthe hyaluronan receptor LYVE-1

Christoph H. Trippa, Bernhard Haida, Vincent Flachera, Michael Sixtb, Hannes Peterc,Julia Farkasc, Robert Gschwentnerd, Lydia Sorokine, Nikolaus Romania,Patrizia Stoitznera,

aDepartment of Dermatology and Venereology, Innsbruck Medical University, Anichstrasse 35, A-6020 Innsbruck, AustriabMax-Planck-Institute for Biochemistry, Martinsried, GermanycDepartment of Microbiology, University of Innsbruck, Innsbruck, AustriadDepartment of Zoology, University of Innsbruck, Innsbruck, AustriaeInstitute for Physiological Chemistry and Pathobiochemistry, Munster University, Germany

Received 23 July 2008; accepted 23 July 2008

Abstract

Langerhans cells and dermal dendritic cells migrate to the draining lymph nodes through dermal lymphatic vessels.They do so in the steady-state and under inflammatory conditions. Peripheral T cell tolerance or T cell priming,respectively, are the consequences of migration. The nature of dendritic cell-containing vessels was mostly defined byelectron microscopy or by their lack of blood endothelial markers. Selective markers for murine lymph endotheliumwere hitherto rare or not available. Here, we utilised recently developed antibodies against the murine hyaluronanreceptor, LYVE-1, to study the lymph vessel network in mouse skin in more detail.

In hairless skin from the ears, lymph vessels were spread out in a horizontal plane. They formed anastomoses, andthey possessed frequent blind endings that were occasionally open. Lymph vessels were wider than blood vessels, whichwere identified by their strong CD31 expression. In body wall skin LYVE-1 reactive vessels did not extend laterally butthey dived straight down into the deeper dermis. There, they are connected to each other and formed a network similarto ear skin. The number and width of lymph vessels did not grossly change upon inflammatory stimuli such as skinexplant culture or tape stripping. There were also no marked changes in caliber in response to the TLR 7/8 ligandImiquimod.

Double-labelling experiments of cultured skin showed that most of the strongly cell surface MHC II-expressing (i.e.activated) dendritic cells were confined to the lymph vessels. Langerin/CD207+ cells within this population appearedlater than dermal dendritic cells, i.e. langerin-negative cells. Comparable results were obtained after stimulating theskin in vivo with the TLR 7/8 ligand Imiquimod or by tape stripping.

In untreated skin (i.e. steady state) a few MHC II+ and Langerin/CD207+ cells, presumably migrating skindendritic cells including epidermal Langerhans cells, were consistently observed within the lymph vessels. The novel

e front matter r 2008 Elsevier GmbH. All rights reserved.

bio.2008.07.025

TLR, toll-like receptor.

ing author.

ess: [email protected] (P. Stoitzner).

s article as: Tripp, C.H., et al., The lymph vessel network in mouse skin visualised with antibodies against the hyaluronan receptor

unobiology (2008), doi:10.1016/j.imbio.2008.07.025

www.elsevier.de/imbiodx.doi.org/10.1016/j.imbio.2008.07.025mailto:[email protected]/10.1016/j.imbio.2008.07.025

ARTICLE IN PRESSC.H. Tripp et al. / Immunobiology ] (]]]]) ]]]]]]2

antibody reagents may serve as important tools to further study the dendritic cell traffic in the skin under physiologicalconditions as well as in conditions of adoptive dendritic cell transfer in immunotherapy.r 2008 Elsevier GmbH. All rights reserved.

Keywords: Dendritic cell migration; Inflammation; Lymphatic vessels; Steady-state migration

Introduction

Dendritic cells of the skin, i.e. epidermal Langerhanscells and dermal dendritic cells (Valladeau and Saeland,2005; Romani et al., 2008), initiate immune responsesagainst antigens/pathogens that enter the body throughthe skin. Thus, they serve as sentinels of the immunesystem. Only a few recent examples exist of innateimmune responses carried out by skin dendritic cells, inparticular Langerhans cells (De Witte et al., 2007). Incontrast, ample evidence exists about the ability of skindendritic cells to induce adaptive immune responses(Romani et al., 2006), contact hypersensitivity being theclassical example for this (Streilein and Bergstresser,1984). Several important questions are not yet answered,though. First of all, as opposed to the well-definedepidermal Langerhans cell, dermal dendritic cells arestill not unequivocally characterised. Subsets may exist(Angel et al., 2006; Bursch et al., 2007; Ginhoux et al.,2007; Poulin et al., 2007), and a transformation fromresident macrophages (Dupasquier et al., 2004; Zabaet al., 2007) into dermal dendritic cells upon organculture or in vivo was recently observed (Dupasquieret al., 2008). The relative contributions of Langerhanscells versus dermal dendritic cells are just beginning tobe understood, thanks to the advent of mouse modelsthat selectively deplete Langerhans cells (Bennett et al.,2005; Kaplan et al., 2005; Kissenpfennig et al., 2005).Surprisingly, Langerhans cells may be less immungenicin vivo, perhaps even down-regulatory (Kaplan et al.,2005), as anticipated from some of the in vitro data.Furthermore, it is not entirely clear as to how skindendritic cells contribute to the maintenance of periph-eral tolerance (Mayerova et al., 2004; Shibaki et al.,2004). Langerhans cells and/or dermal dendritic cellscan constitutively capture self-antigens from the skinand carry them to the peripheral lymph nodes (Hemmiet al., 2001). As long as this happens in the steady state,peripheral tolerance is sustained (Steinman and Nus-senzweig, 2002). The relative contributions of Langer-hans cells and dermal dendritic cells in this process havenot yet been elucidated, though.

Irrespective of these uncertainties about the behaviourof skin dendritic cells, there is one precondition thatneeds to be fulfilled at any rate. Dendritic cells mustmigrate from the skin to the draining lymph nodes, i.e.those sites where primary immune reactions areinitiated, be they immunogenic or tolerogenic in nature.Many conditions under which dendritic cell migration

Please cite this article as: Tripp, C.H., et al., The lymph vessel network in m

LYVE-1. Immunobiology (2008), doi:10.1016/j.imbio.2008.07.025

takes place have been described in the past. Migration isregulated at the levels of adhesion, enzymatic digestionof the extracellular matrix, chemotaxis to chemokines(Romani et al., 2001; Randolph et al., 2005) and byspecialised conduit systems in the lymph nodes (Sixtet al., 2005). The necessary signals are induced byinflammatory cytokines and toll-like receptor (TLR)ligands as well as physical stimuli. Various TLR ligandshave been described as potential modulators of the skin.Ligation of TLRs induces the production of inflamma-tory cytokines and chemokines as well as maturationand migration of DC. Recent data from mouse modelsstrongly suggested that DC migration can be improvedby treating skin with proinflammatory cytokines orTLR 7 ligand Imiquimod (Nair et al., 2003).

Comparably few data are available about the routesof dendritic cell migration, mostly the lymph vessels ofthe skin (reviewed in Angeli et al., 2006). Not least, thiswas due to the fact that antibodies recognising lymphvessels have not been available for long (Breiteneder-Geleff et al., 1999; Gale et al., 2007). This prompted usto study in more detail the lymph vessel network inmouse skin and to better define dendritic cells travellinginside these vessels. This was achieved by using anantibody against lymphatic vessel endothelial hyalur-onan receptor-1 (LYVE-1), exclusively expressed onlymphatic endothelial cells in mouse dermis (Banerjiet al., 1999; Prevo et al., 2001). LYVE-1 is a receptor forhyaluronan, a carbohydrate component of the cuta-neous extracellular matrix, presumably involved in theregulation of interstitial-lymphatic flow (Huang et al.,2006), lymphangiogenesis (Chen et al., 2005), andpossibly also in innate immune functions (Bockleet al., 2008).

We focused on the morphology and spatial orienta-tion of lymphatic vessels in murine skin. Furthermore,we investigated the occurrence and proportions of skindendritic cells within lymphatic vessels of inflamed andsteady-state skin, with the aim to gain more insights intothe regulation of dendritic cell migration.

Materials and methods

Mice and media

Mice of inbred strains C57BL/6 and BALB/c werepurchased from Charles River Laboratories (Sulzfeld,

ouse skin visualised with antibodies against the hyaluronan receptor

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Germany) and used at 28 months of age. Allexperimental protocols were approved by the AustrianAnimal Ethics Committee and performed according toinstitutional guidelines. The culture medium used forexplant cultures was RPMI-1640 supplementedwith 10% heat-inactivated FCS (Biochrom, Berlin,Germany), 2-mM L-glutamine (Sigma Chemicals,St. Louis, MO), 50-mg/ml gentamycin (PAA, Linz,Austria), and 50-mM 2-mercapto-ethanol (Sigma).

Skin explant culture

This was based on established methods (Romaniet al., 1997). Ear skin from mice was split into dorsaland ventral halves, and the dorsal (i.e. cartilage-free)halves were cultured in 24-well tissue culture plates (oneear per well) for 2496 h.

In vivo treatment of skin

Tape stripping was performed as previously described(Holzmann et al., 2004). Briefly, ears were stripped10 times with ordinary adhesive tape (TIXOs, made inUSA; TransporeTM surgical tape, 3M Health Care,St. Paul, MN). For each stripping a fresh piece of tapewas lightly pressed onto the ear and pulled off. TLRligand Imiquimod was used in its pharmaceutical form,AldaraTM cream (5% Imiquimod; 3M Pharmaceuticals,St. Paul, MN, USA) and applied topically to the ears.

Preparation of dermal sheets forimmunofluorescence analyses

After defined time points mice were sacrificed andepidermal and dermal sheets prepared as described withminor modifications (Juhlin and Shelley, 1977). In brief,ears were cut off, dorsal halves were separated from thecartilage-containing ventral ear halves and floatedepidermal side up on 0.5-M ammoniumthiocyanatesolution for 20min at 37 1C. Epidermis and dermis wereseparated, cut into small pieces and fixed in acetone or2% paraformaldehyde for 1020min at room tempera-ture. Samples were washed twice for 20min in PBS andPBS/1% BSA and then used for fluorescent labelling.

Antibodies and immunofluorescence protocols

The following anti-LYVE-1 antibodies were used: apolyclonal rabbit antiserum was produced by LydiaSorokin, Munster, Germany (then Lund, Sweden);another polyclonal rabbit Ig anti-murine LYVE-1(immunogen: amino acids 267284) was purchased fromUpstate Biotechnology Inc. (Lake Placid, NY, USA). Inaddition, rat anti-CD31mAb (clone MEC 13.3;IgG2a; BD Biosciences, San Diego, CA, USA), rat



anti-I-Ad/I-Ed mAb (clone 2G9; IgG2a; FITC-conju-gated, BD), rat anti-I-Ab,d (clone B21-2; IgG2b;hybridoma supernatant), rat anti-CD86 (clone GL1;IgG2a, BD), and anti-murine Langerin/CD207 (clone929.F3; rat IgG1; Dendritics, Lyon, France) wereapplied. Isotype-matched control immunoglobulinswere purchased from DAKO (Glostrup, Denmark).Rat monoclonal antibodies were visualised by biotiny-lated anti-rat Ig followed by Streptavidin-Texas Red(both from Amersham Biosciences, Amersham, UK).Counterstaining for MHC II was done with FITC-conjugated anti-I-Ad/I-Ed after the preceding blockingof residual free-binding sites with an excess of rat Ig(100 mg/ml, 15min). Rabbit anti-LYVE-1 was detected(i) by a biotinylated donkey anti-rabbit Ig followed byStreptavidin-FITC (both from Amersham Biosciences)or (ii) by an Alexa 488-conjugated cross-absorbed(against rat Ig) goat anti-rabbit Ig antibody (Invitrogen,Molecular Probes), or (iii) by an FITC-conjugated swineanti-rabbit Ig (DAKO). This choice of green fluoro-chromes allowed counterstaining with rat Ig anti-Langerin followed by a chicken anti-rat IgG (H+L)antibody conjugated to Alexa 594 (Invitrogen-Molecu-lar Probes, Cat. No. A21471). Non-specific backgroundwas minimised by using the Image iTTM reagent kitfrom Invitrogen according to the manufacturers proto-col. Specimens were viewed on a conventional OlympusBX60 epifluorescence microscope equipped with adigital camera (Olympus) for documentation. Selectedspecimen were inspected with an AxioImager.Z1 epi-fluorescence microscope (Zeiss, Oberkochen, Germany),equipped with a high-resolution colour camera (Axio-cam HRC, Zeiss). High-resolution overview pictures(Fig. 1J) were automatically taken using Autofocus- andMosaiX function of the AxioVision software (Zeiss).

Results

Organisation of the lymphatics in ear and body wallskin

Dermal sheets were stained with rabbit Ig anti-LYVE-1antibody. In the nearly hairless skin from ears the lymphvessels were spread out in one horizontal plane (Fig. 1Dand J). They formed irregular anastomoses, and theypossessed frequent blind endings that were occasionallyopen (Fig. 1E and F). Lymph vessels were consistentlywider than blood vessels that were clearly distinguishedby their CD31 expression (Fig. 1AC). In contrast, inbody wall skin LYVE-1 reactive vessels did not extendlaterally but they were homogenously distributed anddived vertically down into the deeper dermis (Fig. 1Gand H) where they connected to each other and formeda horizontal network similar to that seen in ear skin


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Fig. 1. Dermal lymphatic vessels in ear and body wall skin. Dermal sheets were stained with anti-CD31 or rabbit polyclonal anti-

LYVE-1 antibody as indicated. CD31 is expressed by blood vessels in ear (A) and body wall dermis (B, C). LYVE-1 is expressed by a

network of lymphatic vessels in ear skin (DJ) or in vertical endings of lymphatic vessels in body wall skin (GI). Note the open

ending of a body wall vessel (E, arrow, and I). Original magnifications were 25 for pictures (A, B, D, G); 100 for (C, E, F, H)and 250 for I. Pictures are representative for one out of four experiments. The bottom panel (J) of this figure represents a view ofan entire ear half, consisting of four individual sheets. The sheets were photographed with the confocal scanning microscope at high

power and subsequently assembled by computer software. Diameter of the ear is approximately 1 cm.

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(data not shown). Furthermore, high magnificationsconfirmed that subepidermal endings of lymphaticvessels of body wall dermis formed open funnels(Fig. 1I).



In summary, the skin is drained by a tight vascularnetwork of subepidermal LYVE-1-positive vessels.Importantly, however, our data show that thethree-dimensional layout and morphology of dermal


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lymphatic vessels are not uniform across the body butare strictly dependent on the skin site.

Dendritic cells migrate within LYVE-1-positivevessels in the steady state

We wanted to evaluate whether cutaneous dendriticcells do migrate via lymphatic vessels in the steady-stateenvironment of skin as implicated by recent findingsthat melanin particles are constantly trafficked to lymphnodes by skin dendritic cells (Hemmi et al., 2001;Yoshino et al., 2006).

By double-labelling untreated dermis of ear skin forLYVE-1 against MHC-II, CD86 and Langerin/CD207,we were able to identify numerous, mostly singletravelling MHC-II+ cells within LYVE-1+ vessels(Fig. 2, row B). It is noteworthy that we never detectedlarge clusters (cords) of stained cells in lymphaticvessels of steady-state skin as compared to skin explantcultures. Occasional groups of a few adjacent intravas-cular MHC-II+ cells were found, however. They mayperhaps represent LC travelling in response to minuteinflammatory stimuli in the epidermis (e.g. minorscratching stimulus). In comparison to MHC-II+ cells,only rare Langerin+ (Fig. 2, row C) or CD86+ cells(Fig. 2, row A) could be detected in lymphatic vessels.

Fig. 2. Migrating dendritic cells in lymphatic vessels of steady-stat

column, green) and MHC-II, CD86 and Langerin/CD207 (middle

depicted in the right column. A single CD86-positive cell, within a

shows two MHC-II+, the lower row a single CD207+ cell in a lym

experiments.



MHC-II+ cells were at least two or three times as manyas Langerin+ or CD86+ cells (data not shown) withinLYVE-1+ lymphatic vessels of steady-state skin. Thus,the frequency of Langerin+ or CD86+ cells was notcomparable to the frequency of MHC-II+ cells. This isalso underlined by the fact that intralymphaticallydetected cells were heterogenously distributed in allinvestigated skin samples.

From these findings we conclude that differentsubtypes of cutaneous DC migrate intralymphaticallyin a non-inflammatory skin milieu. Langerhans cells andLangerin+ dermal dendritic cells represent a minorproportion of these cells.

Dendritic cells migrate within LYVE-1-positivevessels under inflammatory conditions

In the experiments described above we investigatedthe migration of DC in lymphatic vessels of untreatedskin. Here, we compare those findings with DCmigration in a model of inflammatory skin environment.Explants from ear skin were cultured for 23 days anddermal sheets were then immunolabelled with antibodiesagainst LYVE-1 and MHC-II or, alternatively, Langer-in/CD207 and CD86. As previously described (Larsenet al., 1990; Stoitzner et al., 2003), many typical cords,

e skin. Freshly prepared dermis was stained for LYVE-1 (left

column, red). Double exposures of both fluorochromes are

LYVE-1+ vessel, is shown in the upper row. The middle row

phatic vessel. Pictures are representative of one out of three


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Fig. 3. Migrating dendritic cells in cultured skin explants. Dermal sheets from skin explants on d2 of culture were stained for LYVE-

1 (red fluorescence) and MHC II (green fluorescence) depicted in AC and row D. Migrating dendritic cells express high levels of

MHC II. This becomes unequivocally evident in an optical traverse section through one Lyve-1+ lymph vessel from body wall skin

(C). Dermal sheets from skin explants on d2 of culture were stained for Langerin/CD207 (row E) and CD86 (row F) in red

fluorescence and for LYVE-1 in green fluorescence. Double exposures of both colours are depicted in the right columns of rows DF.

Conventional fluorescence microscopy (A, B and rows DF), confocal microscopy (C). Scale bar in (C) corresponds to 10 mm.Pictures are representative of one out of three experiments.

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i.e. accumulations of strongly MHC II+ cells in a string-like fashion, were found. These cells were surrounded byLYVE-1+ lymphatic vessels (Fig. 3A and row D). Thelevel of LYVE-1 expression as well as vessel morphologywas unchanged compared to steady-state dermis. Evenin conventional fluorescence microscopy it becameapparent that migrating dendritic cells displayed fea-tures of maturity such as high expression of MHC II anda distinct veiled or hairy morphology (Fig. 3B). Byfocusing on vertical lymphatic vessels of body walldermis we could demonstrate that cells were within theopen lumen of LYVE-1+ vessels and MHC-II wastranslocated to the cell surface (Fig. 3C, confocalmicroscopy), suggesting that those migrating DC are,at least partially, mature (Pierre et al., 1997). Ascompared to steady-state dermis, more Langerin+

(Fig. 3, row E) and CD86+ cells (Fig. 3, row F) were



found in the lumen of LYVE-1+ vessels as well asdispersed in the dermal connective tissue (data notshown). Like in untreated skin, blood endothelial cellswere never positive for LYVE-1 in inflamed skinsamples. This could be concluded from the observationthat LYVE-1 expression was never detected on vascularstructures with a typical thin blood vessel morphology(see Fig. 1AC).

To further extend these findings, we tried to assess theproportions of Langerhans cells (i.e. Langerin+/MHCII+)and dermal dendritic cells (i.e. Langerin/MHCII+

cells) within the lymph vessels in skin explant culturesafter 24, 48, 72 and 96 h of culture. This was done in asemi-quantitative way by simply counting the differentcell types in Langerin and MHC-II double-labelleddermal sheets. In this case, lymph vessels were notidentified by their LYVE-1 expression but simply on the


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Fig. 4. Proportions of Langerhans cells and dermal dendritic cells in dermal lymphatic vessels. Dermal sheets of skin explants,

cultured for 24, 48, 72 and 96 h, were double labelled for Langerin/CD207 (red fluorescence, left column) and MHC-II (green

fluorescence, middle column). Double exposures of both fluorochromes are depicted in the right columns. The fraction of CD207+/

MHC-II+ Langerhans cells within vessels reaches a maximum at 48 h of culture. Pictures are representative of one out of three

experiments. Sixteen epidermal sheets were analysed per time point.

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basis of the unequivocal shape of these strings (cords) ofbrightly fluorescing cells (Fig. 4) (Stoitzner et al., 2003).As previously described, at time points 48 and 72 h(Fig. 4, middle rows) (i.e. the peak of cord formation inperformed explant cultures) the proportions varied fromcord to cord from a majority of dermal DC to a majorityof Langerhans cells. Overall, similar numbers of bothtypes of dendritic cells migrated within the cords at thesetime points. Of note, increasing numbers of interstitial,non-cord-associated, Langerhans cells could be seen at48 h of culture. At the early time point of 24 h, however,there was a clear preponderance of dermal dendritic cells(Fig. 4, upper row). In the few cords that could bedetected at this time point, clusters of MHC-II+/Langerin-negative cells were observed. Only sporadi-cally could Langerin+/MHC-II+ cells be spotted.Interestingly, the proportion of Langerhans cells de-creased again at the 96-h time point, and so did thefrequency of observed cords (Fig. 4, lower row).



These results underline and extend recent findings thatcutaneous DC populations concentrate and migratewithin dermal lymphatic vessels of inflamed skin.

Accordingly, MHC-II+ dermal dendritic cells migratemostly in the first 24 h of culture followed by atemporally delayed population of Langerin+/MHC-II+

dendritic cells. We could observe that the maximum ofdermal, intralymphatic DC migration takes placebetween 48 and 72 h of culture.

Dermal lymphatics do not grossly change uponstrong inflammatory stimuli

Next, we were wondering whether the number or thestructure of dermal lymphatics would change in aninflammatory milieu elicited differently from the above-mentioned skin explant cultures. We applied tapestripping, a mechanical stimulus that does not involve


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an exogenous TLR ligand. For comparison, we inflamedthe skin by the topical application of Imiquimod cream,a ligand for TLR7/8. Forty-eight hours after theapplication of these stimuli, dermal sheets were stained.Epidermal sheets served as a positive control. Inresponse to either treatment, the numbers of Langer-hans cells in the epidermis dropped and the remainingLangerhans cells appeared activated, as described(Suzuki et al., 2000; Holzmann et al., 2004; Heibet al., 2007). This assured that the inflammatorystimulus was indeed effective (data not shown). Theappearance of the lymphatic network did not grosslychange in response to the inflammation stimuli. Num-bers of lymph vessels were similar and their calibers were

Fig. 5. Morphology of dermal lymphatics does not change upon in

(green fluorescence) 48 h after topical treatment of skin with Imiquim

cells (row A). Expression of LYVE-1 is reduced after tape stripping.

(red fluorescence) is depicted for Imiquimod (panel B) and tape stripp

depicted in the right columns of panels B and C. Pictures are repre



also unchanged (Fig. 5, row A). As previously described(Johnson et al., 2006, 2007), we also observed somereduction in the intensity of LYVE-1 staining uponthese treatments, in particular in response to tapestripping (Fig. 5, row A). This was not uniform, though.There were always distinct areas with apparentlyunchanged staining intensity (data not shown).

To further elucidate whether those inflammatorystimuli could have an impact on intralymphaticDC migration, we double-labelled dermal sheets forLYVE-1 and MHC-II or Langerin/CD207. There wereclearly more MHC-II+ cells visible in lymphatic vesselsof tape-stripped skin (Fig. 5, panel C) compared toImiquimod-treated (Fig. 5, panel B) or untreated skin

flammatory stimuli. Dermal sheets were stained for LYVE-1

od or tape stripping. Red double-labelling identifies MHC-II+

Additional double labelling for MHC-II and Langerin/CD207

ing (panel C) treatment. Double exposures of both colours are

sentative of one out of two experiments.


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(see Fig. 2). Remarkably, in contrast to skin explantculture, only a small proportion of the MHC-II+ cellsappeared positive for Langerin 48 h after application ofthe inflammatory stimulus.

Our observations support the recent findings thatinflammatory stimuli, such as tape stripping, couldreduce the levels of LYVE-1 expression on dermallymphatic vessels. Additionally, tape stripping of theskin appeared to be more potent in activating DCmigration compared to the application of the TLRligand Imiquimod.

Discussion

In this study, we describe the expression of LYVE-1on dermal lymphatic vessels of mouse skin. Firstly, weobserved conspicuous differences in the lymph vesselnetwork between body wall skin and ear skin. Secondly,we observed a down-regulation of LYVE-1 expressionin the dermis of inflamed skin. Thirdly, we demonstratemigratory dendritic cells within lymphatic structures inan inflammatory and non-inflammatory (steady state)skin environment. Few dendritic cells travelled throughthe lymph vessels in the steady state whereas underinflammatory conditions we observed numerous, intra-lymphatic, mainly mature dendritic cells. The fraction ofLangerhans cells peaked at a delayed time period ascompared to dermal dendritic cells. Finally, we couldnot detect marked changes in the quantity andmorphology of dermal lymph vessels in response toinflammatory stimuli.

Anatomical differences in the structure of thelymphatics

The present data confirm and extend recent findingsthat in the skin, murine LYVE-1 is uniquely expressedon lymphatic endothelial cells and absent from bloodendothelial cells as well as from non-vascularised tissues,such as epidermis (Banerji et al., 1999; Prevo et al., 2001;Cueni and Detmar, 2006). The function of the hyalur-onan receptor LYVE-1 is still disputed: LYVE-1 wassuggested to mediate the transport of hyaluronan out ofskin and govern the contact and transport of dendriticcells into and through lymphatic vessels (Jackson,2004; Chen et al., 2005). However, recent studies withLYVE-1 knockout mice revealed that hyaluronantransport and dendritic cell traffic is independent ofLYVE-1 (Huang et al., 2006; Gale et al., 2007). Mostrecently, Johnson et al. (2007) found that LYVE-1 isreversibly down-regulated under inflammatory condi-tions, suggesting a function for cellular traffic uponinfections and chronic skin disease.



Our experiments revealed that ear and body wall skinappeared to be differently drained by the lymphatics.One can only speculate about a possible biologicalrelevance of this finding. Body wall skin carries a thickfur, whereas ear skin is almost hairless and lacks apronounced fat droplet-filled subcutis that could pro-vide space for collecting deep-dermal lymphatic ducts.Therefore, we suppose that mouse body wall skinreflects the lymphatic architecture in human skin betterthan ear skin. In addition, hairless skin, such as ear skin,is certainly more exposed to pathogenic threats as wellas mechanical irritation and it may therefore require adenser and more efficient system of draining lymphaticsas opposed to body wall skin that can fend off intrudersby the dense hair coat. The experimental model of skinexplant cultures seems to support this assumption.Migratory dendritic cells (so-called crawl-out dendri-tic cells) can best be obtained from explants of ear skin.All attempts to reach comparable numbers of migrantdendritic cells from whole-skin explants of murine bodywall skin failed; only very rare cells emigrate from theseexplants (Ortner et al., 1996).

The here-observed differences in the lymphatics maybe an explanation for this phenomenon. In this regard itwould be interesting to compare the cutaneous lympha-tics of hairless (i.e. normal) versus hairy (i.e. scalp)human skin.

Dendritic cell populations of the dermis

The main DC population are Langerin-negativedermal dendritic cells (Lenz et al., 1993). There is alsoa minor fraction of Langerin+ cells in the dermis. Thesecells were generally assumed to be epidermal Langer-hans cells in transit through the dermis. Three recentpublications changed the picture. A novel population ofnon-epidermis-derived, radio-sensitive Langerin+

dermal DC was found and characterised (Burschet al., 2007; Ginhoux et al., 2007; Poulin et al., 2007).We would like to emphasise that in our manuscript, forthe sake of simplicity, the term Langerhans cells in thecontext of dermis and dermal vessels comprises bothepidermis-derived Langerhans cells in transit and thenovel Langerin+ population. To date, it is not possibleto unequivocally distinguish these populations fromeach other in situ by immunofluorescence. In dermal cellsuspensions, CD103 expression, as determined by flowcytometry, appears to be a useful marker for the novelpopulation (Bursch et al., 2007).

Dermal lymphatics under steady-state conditions

Our observations demonstrate that subtypes ofskin dendritic cells migrate within dermal lymphaticvessels from non-inflammatory, i.e. steady state, skin.


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Lymphatic vessels from unaffected skin containednumerous MHC-II+ cells. MHC-II/Langerin double-positive Langerhans cells or phenotypically matureMHC-II+/CD86+ dendritic cells reflected a smallfraction of migrating cells.

Dendritic cells critically contribute to the inductionand maintenance of specific peripheral tolerance(Steinman and Nussenzweig, 2002). For this purpose,dendritic cells were supposed to constitutively migrate ina rather immature state from peripheral tissues todraining lymph nodes, presenting self-antigen (Steinmanand Nussenzweig, 2002). Indeed, skin dendritic cells, inparticular Langerhans cells, were previously shownto capture self-antigen in the steady-state skin and totransport it to the draining nodes (Hemmi et al., 2001;Yoshino et al., 2006). These Langerhans cells traffickingfrom steady-state epidermis are uniquely replenished byslowly self-renewing precursors within the epidermis(Merad et al., 2002). Whether it is those migrating andself-antigen-carrying skin dendritic cells that directlyinduce peripheral tolerance by T cell deletion orinduction of regulatory T cells (Hawiger et al., 2001;Methe et al., 2007) is not clear.

Regarding the question of how dendritic cell migra-tion and presentation is managed during steady state, itbecame recently evident that migrating dendritic cells inthe steady state, i.e. lacking inflammatory stimulation,are not immature. They express some phenotypical signsof maturity such as CD40 and CD86 when analysed inlymph nodes (Ohl et al., 2004; Stoitzner et al., 2005;Waithman et al., 2007). Wilson et al. (2008) observedthat even in germ-free mice the lymph node populationof migratory dendritic cells is as mature by phenotypicalcriteria as in normal mice. This suggests an indepen-dence of steady-state dendritic cell traffic from patho-gen-triggered TLR signalling, although a mechanicallyinduced inflammation of skin areas may not be excludedin this regard. In support of this, Ohl et al. (2004)described that the expression of chemokine receptorCCR7, decisive for dendritic cells to enter lymphaticvessels, is essential for the steady-state migrationof Langerhans cells to lymph nodes. Moreover, theCCR7 ligand CCL21 was shown to be expressed bynonstimulated lymphatic endothelial cells in vitro(Kriehuber et al., 2001) and lymph vessels of unaffected,healthy human (Eberhard et al., 2004) and murine(Saeki et al., 1999) skin. Therefore, consideringthe rather poorly defined phenotype of steady-statetrafficking dendritic cells (and the fraction ofCD86+ dendritic cells thereof that we observedin our study) we have to concede that it cannot beunequivocally determined whether these cells wereforced into migration by locally small microinflamma-tory stimuli or whether they were indeed thosecontinually trafficking dendritic cells inducing periph-eral tolerance.



From our observations we conclude that subtypes ofcutaneous dendritic cells migrate intralymphatically inthe steady state, presumably utilising similar mechan-isms as migrating dendritic cells do under inflammatoryconditions. The proportions, detailed phenotypes andpotentially variable functions of those steady-statedendritic cell subtypes remain to be clarified.

Dermal lymphatics under inflammatory conditions

Larsen et al. (1990) were the first to describe theconspicuous accumulation of migrating skin dendriticcells in the so-called cords. These structures were latercharacterised as lymphatic vessels by electron micro-scopy (Weinlich et al., 1998; Stoitzner et al., 2002). Here,we extend these findings by ascertaining that theselymph vessels express the LYVE-1 receptor.

Our observation that in the early phase of skinexplant cultures (i.e. day 1) most migrating cells withinthe lymph vessels were dermal dendritic cells, ratherthan Langerhans cells, fits well with observations byKissenpfennig et al. (2005) who observed in vivo thatdermal dendritic cells arrived in the draining lymphnodes earlier than Langerhans cells.

In a second set of experiments we treated ear skin withTLR 7/8 ligand Imiquimod or with tape stripping. Veryrecent reports proved that topically applied Imiquimodinduced migration of skin-resident DC via mast cells(Heib et al., 2007). Mechanical stress applied to skin,such as tape stripping, induced significant enhancementof DC migration (Holzmann et al., 2004). Accordingly,we were able to detect numerous migrating dendriticcells in the lymphatic vessels subsequent to the above-mentioned in vivo skin stimulation. Activated, phenoty-pically mature skin dendritic cells express CCR7 (Ohlet al., 2004) and hence respond to its ligand CCL21,which is expressed by lymphatic endothelial cells (Saekiet al., 1999; Eberhard et al., 2004). Furthermore, theexpressions of CXCR4 on cutaneous dendritic cellsand of the adequate ligand CXCL12 on lymphaticvessels are pivotal for dendritic cell migration(Kabashima et al., 2007). The leucocyte adhesionreceptors intercellular adhesion molecule 1 (ICAM-1)and vascular cell adhesion molecule 1 (VCAM-1) werealso shown to be induced in lymph vessels of inflamedskin and regulate dendritic cell traffic as well (Johnsonet al., 2007).

Although the basic mechanisms for the migration ofmature dendritic cells seem to be understood, there issome dissonance on how the congruent signals find eachother via the afferent lymphatics. Since there is acontinuous lymph flow from skin to the lymph node(making the hypothesis of simple chemokine gradientssomewhat implausible), it remains unclear as to howactivated dendritic cells can follow a signal that exists


dx.doi.org/10.1016/j.imbio.2008.07.025


within the lymph vessel or node (Randolph et al., 2005;Angeli et al., 2006). On the other hand, chemokinegradients created by interstitial flow may help migratingskin dendritic cells to find their way into lymph vessels.This novel concept was termed autologous chemo-taxis by M. Swartz (Reddy et al., 2007; Shieldset al., 2007). It implies that chemokines such asCCL19/MIP-3, which can indeed be made by mature(migrating) dendritic cells (Hofer et al., 2004), getsucked away from the producing cells towards thelymph vessels by interstitial flow. This creates a gradientthat can be sensed and followed by CCR7 on the samemigrating dendritic cells (reviewed in Lammermann andSixt, 2008). The efficiency of dendritic cell migration isfurthermore increased by the recently discovered factthat it occurs independent of (possibly slowing) interac-tions with the integrins of the extracellular matrix(Lammermann et al., 2008).

Nevertheless, most of the above-mentioned signals,which were first observed in inflammatory skin, are alsopresent in steady-state skin, albeit at low levels. In fact,the expression of CCR7 (Ohl et al., 2004), CCL21(Kriehuber et al., 2001; Eberhard et al., 2004) andCXCL12 (Kabashima et al., 2007) was shown foruntreated skin. Our data evidence that dendritic cells,starting from inflammatory or non-inflammatory skin,migrate via the same afferent lymphatic path.

Biological relevance

The in vivo function of LYVE-1 is still disputed;presumably it regulates lymphatic cellular traffic underinflammatory conditions (Johnson et al., 2007). Expres-sion of LYVE-1 on lymphatic vessels was extensivelyinvestigated in association with tumour-dependentlymphangiogenesis and metastasis. In the case ofmalignant melanoma, several groups published thatmelanoma cells induce new lymphatic vessels in oraround tumours and subsequently metastasise throughthem. Yet, it appeared not feasible to use LYVE-1expression as a prognostic factor (Straume et al., 2003;Giorgadze et al., 2004; Dadras et al., 2005; Sahni et al.,2005).

Dendritic cells loaded with tumour antigens havebeen widely applied in clinical trials, e.g. for theimmunotherapy of mostly malignant melanoma. In thiscase, the preferred pathway of administration is theskin. In most cases, dendritic cells are injected intrader-mally. It is expected that as many injected cells aspossible enter the lymphatics and travel to the drainingnodes in order to induce immunity against the tumour.It has repeatedly been shown that this is indeed the case(Banchereau and Palucka, 2005; Enk et al., 2006;Gilboa, 2007). On the other hand, however, it has alsobeen shown that the migration of dendritic cells placed



into the dermis is very inefficient. Only small percen-tages of injected cells make it all the way into the lymphnodes (Nair et al., 2003). This was shown in mice andalso in human volunteers by means of radioactivelymarked cells (Morse et al., 1999; Blocklet et al., 2003; DeVries et al., 2003; Nair et al., 2003). Therefore, it isdesirable to improve dendritic cell migration (Ademaet al., 2005). Preconditioning of the skin withpre-injected dendritic cells has been suggested (Martn-Fontecha et al., 2003). It has not proven feasible yet inthe clinical context, though. The application of inflam-matory mediators, including TLR ligands, to the skinmay offer more practical alternatives. Our own ongoingexperiments in mouse models do not yet show a markedimprovement of migration (unpublished observations)and the data presented here do not show an influence of(admittedly only three) inflammatory stimuli on num-bers and calibers of dermal lymph vessels.

Based on the findings described here we propose tofurther monitor the behaviour of the lymph vesselnetwork in response to inflammatory stimuli as anadditional screen for finding optimal conditions fordendritic cell migration, but just as well to obviatemetastatic spread. Remarkably, recent studies suggestthat lymphatic endothelial cells express various TLRs(Kuroshima et al., 2004; Fitzner et al., 2008), thusadding a new participator to cutaneous pathogenresponses. It became obvious that both phenotypicallyimmature (Geissmann et al., 2002) and mature (at leastby phenotypical criteria) dendritic cells migrate vialymphatics. These studies, however, did not addresswhether immunity or tolerance was induced. Therefore,one may assume that only certain combinations ofinflammatory stimuli do induce immunity. Notably,Jiang et al. (2007) showed very recently that dendriticcells, matured by mechanical agitation only, exhibitedmigratory capacity without producing inflammatorycytokines or efficient T cell responses. It remains to beseen whether in real life mechanical strain of the skinmay be a trigger for steady-state dendritic cell migrationfrom skin.

Acknowledgments

This work was funded by the Austrian Science Fund(FWF project L120-B13 to NR). We are particularlyindebted to P. Fritsch, Head of Department, for hiscontinued support and encouragement. CHT was inpart funded by the Kompetenzzentrum Medizin Tirol(project CEMIT/KMT-03b). Furthermore, thecontinuous support of the Tyrolean ProvincialHospital Company (Tilak Ges.m.b.H.) is greatlyappreciated.


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http://ISBN:978-81-308-0276-3http://ISBN:978-81-308-0276-3dx.doi.org/10.1016/j.imbio.2008.07.025

The lymph vessel network in mouse skin visualised with antibodies against the hyaluronan receptor LYVE-1IntroductionMaterials and methodsMice and mediaSkin explant cultureIn vivo treatment of skinPreparation of dermal sheets for immunofluorescence analysesAntibodies and immunofluorescence protocols

ResultsOrganisation of the lymphatics in ear and body wall skinDendritic cells migrate within LYVE-1-positive vessels in the steady stateDendritic cells migrate within LYVE-1-positive vessels under inflammatory conditionsDermal lymphatics do not grossly change upon strong inflammatory stimuli

DiscussionAnatomical differences in the structure of the lymphaticsDendritic cell populations of the dermisDermal lymphatics under steady-state conditionsDermal lymphatics under inflammatory conditionsBiological relevance

AcknowledgmentsReferences

the lymph vessel network in mouse skin visualised with ... · the lymph vessel network in mouse...

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