Cutaneous CXCL14 Targets Blood Precursors to Epidermal Niches for Langerhans Cell Differentiation

View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Immunity, Vol. 23, 331–342, September, 2005, Copyright ©2005 by Elsevier Inc. DOI 10.1016/j.immuni.2005.08.012 Cutaneous CXCL14 Targets Blood Precursors to Epidermal Niches for Langerhans Cell Differentiation

Patrick Schaerli,1,3 Katharina Willimann,1 environmental antigens (Steinman et al., 2003). This Lisa M. Ebert,1,4 Alfred Walz,2 and Bernhard Moser1,* steady-state DC function involves the continuous up- 1Institute of Cell Biology take and presentation of self-antigens under noninflam- 2 Theodor-Kocher Institute matory conditions for elimination of self-reactive T cells University of Bern and/or for induction of regulatory/suppressor T cells. CH-3012 Bern DCs also exist in two “migratory” states. Tissue-resi- Switzerland dent DCs express receptors for inflammatory chemokines but lack CCR7, whereas mature or skin-emigrant (semimature) DCs express CCR7 for rapid relocation to Summary draining LNs (Sallusto and Lanzavecchia, 2000). DC maturation is not reversible, suggesting that the pool of tissue-resident DCs is continuously replenished Dendritic cells (DCs) are major constituents of periph- from newly generated precursors. Although a certain eral tissues, where they control immunity to foreign and lineage plasticity cannot be excluded, myeloid DCs de- self-antigens. The process of continuous DC renewal velop from myeloid-committed precursors, which origi- under homeostatic conditions is largely undefined. nate from CD34+ hematopoietic progenitor cells (HPCs) Here, we demonstrate that CD14+ DC precursors, either in bone marrow (Shortman and Liu, 2002; Ardavin et al., derived from CD34+ hematopoietic progenitor cells or 2001). It is not clear at present if the variety of DCs in isolated from blood, were attracted by the chemokine peripheral tissues, including interstitial DCs and Lang- CXCL14, which is constitutively produced in healthy erhans cells (LCs), are directly derived from a common skin and other epithelial tissues. In a tissue model we myeloid DC precursor or from a variety of precursor show that human epidermal equivalents profoundly subtypes with distinct tissue-selective homing proper- affected CD14+ DC precursors, including their supra- ties. DCs are extremely rare in blood and constitute dis- basal positioning and survival as well as their differ- tinct types of “committed” DC precursors (Banchereau entiation into Langerhans cell-like cells with potent et al., 2000; MacDonald et al., 2002). Whether they give antigen-presentation functions. Our model assigns rise to tissue-resident DCs or represent tissue-emigrant unprecedented roles to CXCL14 and epidermal tissue DCs is an open question. Cells resembling tissue-resi- as attractant and niche of differentiation, respectively, dent DCs can be generated in vitro from CD34+ HPCs in the renewal of Langerhans cells under steady- or blood monocytes (Caux et al., 1996; Sallusto and state conditions. Lanzavecchia, 1994). In addition, blood monocytes may develop in situ into DCs (and macrophages) under in- Introduction flammatory conditions (Randolph et al., 1999). How- ever, the precursors that develop into tissue-resident Antigen-presenting cells (APCs) initiate immune re- DCs under steady-state condition have not been iden- sponses by presenting MHC bound peptides to T cells tified. carrying the appropriate T cell receptor. DCs are unique Here, we hypothesized that the migration properties among APCs since they efficiently trigger primary im- of DC precursors define their final destination, where mune responses in naive T cells that need adequate differentiation into tissue-resident DCs may occur. Con- costimulation for full engagement (Banchereau et al., sequently, studies aimed at identifying tissue chemo- 2000; Shortman and Liu, 2002; Steinman et al., 2003). kines with selectivity for circulating DC precursors will The vast majority of DCs are present at extravascular reveal valuable information about the site of DC devel- sites, including epithelial tissues, whereas DCs are opment and homeostasis. Recently, we have shown scarce in blood. that the chemokine CXCL14, alternatively called BRAK A hallmark of DC physiology is the functional duality (Hromas et al., 1999; Frederick et al., 2000), BMAC represented by two states of maturation, which are (Sleeman et al., 2000), and MIP-2γ (Cao et al., 2000), tightly linked to tissue homeostasis and inflammation. is constitutively produced in the epidermis of healthy DCs in healthy peripheral tissues are in an “immature” human skin (Kurth et al., 2001). Here we provide new state characterized by potent microbe-sensing as well evidence that the function of CXCL14 is related to the as antigen-capture and processing capabilities. “Ma- homeostasis of cutaneous DC. Our findings support a ture” DCs home to lymph nodes (LNs) and provide model whereby CXCL14 controls the epidermal recruit- + expert antigen-presentation and T cell stimulation func- ment of CD14 DC precursors, which enables their in tions. Besides a proinflammatory role, DCs are instru- situ differentiation into functional LC-like cells under mental in maintaining peripheral tolerance to self and steady-state conditions.

Results *Correspondence: [email protected] 3 Present address: Department of Pathology, Harvard Medical + + School, The CBR Institute for Biomedical Research, Boston, Mas- CXCL14 Targets in Vitro CD34 HPC-Derived CD14 sachusetts 02115. DC Precursors and Blood Monocytes 4 Present address: Ludwig Institute for Cancer Research (Mel- The two major DC subsets in skin are interstitial dermal bourne Branch), Heidelberg, Victoria, 3084, Australia. DCs and epidermal LCs. To address the question of Immunity 332

skin DC development in homeostasis, we examined the responsiveness of potential skin-homing DC precursors to CXCL14, a chemokine with selectivity for human blood monocytes and prominent expression in healthy human skin (Kurth et al., 2001). The following study is based on the paradigm in chemokine research that pre- dicts an intimate relationship between leukocyte migration properties and leukocyte function. Consequently, we proposed that skin, one major site of CXCL14 production, would influence the subset of CXCL14-responsive cells among blood mononuclear cells. First, we generated DC precursors from blood CD34+ HPCs by in vitro culture, resulting in two nonoverlap- ping subsets of CD1a+ (CD14−) and CD14+ (CD1a−)DC precursors and a third subset of CD1a/CD14-double- negative (DN) cells that have also lost CD34 (Figure 1A; Caux et al., 1996). These subsets appeared as early as day 4 of CD34+ HPC differentiation. The CD1a+ subset contains committed LC precursors, whereas the CD14+ cells resemble blood monocytes in their unrestricted potential to differentiate into either macrophages, interstitial DCs, or LCs (Strunk et al., 1996; Jaksits et al., 1999). CXCL14 is highly selective for CD14+ cells as evidenced in a chemotaxis assay (Figure 1A). Migration responses peaked early, between days 6 and 10 of culture, with a net migration of 19.2% ± 2.3% input cells (mean ± SD;n=6)andchemotactic indices (CIs) of >10, and then returned to base level by day 15. In contrast, CD1a+ cells and DN cells were poorly attracted by CXCL14 during the entire CD34+ HPCs differentiation process. This is underscored by the low CIs of 1.3 and 2.6 for CD1a+ and DN cells, respectively, reflecting mi- nor differences between CXCL14-mediated and background migration. Next, we studied the expression of the unknown CXCL14 receptor by flow cytometry using biotinylated

ferentiated in the presence of GM-CSF, TNF-α, SCF, and Flt-3L and gave rise to three subsets of CD1a+, CD14+, and double-negative (DN) cells (inset). The open square depicts the migration response of freshly isolated CD34+ HPCs. The data are representative for six independent experiments. (B) Analysis of CXCL14 receptors on fresh blood CD14+ monocytes, CD3+ T cells, CD19+ B cells, and CD56+ NK cells by flow cytometry. The gray histograms show CXCL14-receptor stainings obtained with Bio-CXCL14, whereas the open histograms depict control stainings with excess of unbiotinylated CXCL14. Data are representative of two experiments. (C) CXCL14-receptor expression on CD34+ HPCs and CD34+ HPC- derived cell subsets. The upper panels show the differentiation pat- terns and the CD14 versus CD1a gating strategy, and the lower panels show the corresponding CXCL14-receptor expression. Note that CD14+ cells at day 8 of culture stained positive for CXCL14- receptors (R1), whereas sorted CD14+ cells after prolonged culture (day 13) ceased to express CXCL14 receptors (inset). Also, recul- ture of sorted DN cells (R2) for another 8 days yielded CD14+ cells (R3) with only low levels of CXCL14 receptors. Data are representative of five experiments. (D) Migration of CD34+ HPC-derived DN, CD14+, and CD1a+ cells at day 8 of culture to 1000 nM CXCL14, 10 nM CCL5, 10 nM CCL2, 300 nM CCL20, 100 nM CCL19, 100 nM SDF-1, or medium alone (control) as measured in a transwell assay on unseparated cells. Figure 1. CXCL14 Targets CD34-Derived CD14+ DC Precursors Note that each subset shows distinct levels of background migra- (A) Transwell migration performed on cultured, unseparated CD34+ tion (black line). The data represent percentage of migrated cells in HPCs in response to 1000 nM CXCL14. CD34+ HPCs were dif- mean values ± SD (n = 6). Error bars show mean ± SEM. Localization and Differentiation of Epidermal DCs 333

CXCL14 as probe. This modification did not affect func- al., 2003). Therefore, chemokines present on blood tion, as assessed by induction of chemotaxis and Ca2+ microvascular endothelial cells may contribute to leu- mobilization in monocytes (data not shown). In control kocyte extravasation, whereas chemokine-producing experiments, biotinylated CXCL14 uniformly stained tissue cells may define the destination of extravasated blood monocytes but not blood T cells, B cells, or NK leukocytes. Initial studies have revealed that CXCL14 cells, as expected from functional studies (Figure 1B; transcripts are abundant in epithelial tissues, notably Kurth et al., 2001). CD34+ HPCs did not express in skin where epidermal keratinocytes and additional CXCL14 receptors, whereas CD14+ DC precursors at dermal cells were shown to be the main producers day 8 were uniformly positive (Figure 1C). Reculturing (Hromas et al., 1999; Frederick et al., 2000; Sleeman et of sorted day 8 CD14+ cells for another 5 days did not al., 2000; Cao et al., 2000; Kurth et al., 2001). We now support further proliferation but instead resulted in demonstrate that CXCL14 protein is associated with CD14+ cells that lost CXCL14 receptors (inset in Figure dermal blood vessels and epidermal keratinocytes of 1C). Also, prolonged culture of sorted DN cells for an- normal human skin (Figure 2A). Antibodies to CD31 other 8 days led to further proliferation and the genera- stain both blood and lymphatic vessels in frozen section of “late” CD14+ cells with borderline levels of tions, whereas antibodies to podoplanin stain preferen- CXCL14 receptors (Figure 1C). These findings are in full tially lymphatic vessels. Therefore, serial section analy- agreement with the transient responsiveness of CD34+ sis reveals prominent CXCL14 positivity in blood HPC-derived CD14+ cells to CXCL14 (Figure 1A) and vessels in the superficial dermal plexus, although re- suggest that this chemokine targets CD14+ cells that duced CXCL14 positivity was also occasionally ob- have newly developed from CD34+ HPCs. served in lymphatic vessels. CXCL14 is most abundant At day 8 of culture, all three CD34+ HPC-derived sub- in the epidermis, where it is associated with basal kera- sets responded well to a selection of additional chemo- tinocytes as well as more differentiated keratinocytes in kines with known function in DCs and revealed distinct, suprabasal location. Epidermal expression of CXCL14 in part mutually exclusive migration profiles (Figure 1D). appears much more prominent than any other chemo- DN cells responded well to CXCL12 (CI = 14.9), a chem- kine with reported constitutive expression at this site okine with strong activity for CD34+ HPCs and B cells (Schaerli et al., 2004; Ebert et al., 2005), including (Moser et al., 2004), but failed to respond to other CCL1, which targets skin-selective immune surveil- chemokines. CD1a+ cells showed increased background lance T cells, or CXCL12 and CCL20 with broader tar- migration and responded to CCL20 (CI = 4.3), as pre- get cell selectivity. Obviously, epidermis needs to be viously reported for LC precursors (Greaves et al., considered as a major destination of CXCL14-respon- 1997), and to a lesser extent to CCL5. In clear contrast, sive cells. In support of the CXCL14 protein data, CD14+ cells displayed highest responses to CXCL14 freshly isolated keratinocytes and dermal adherent and to CCL2, which is the prototype monocyte chem- cells, including fibroblasts and endothelial cells, ex- oattractant. Finally, CD14+ cells but not DN and CD1a+ pressed CXCL14 mRNA, as evidenced by Northern blot cells expressed monocyte-typical cell-surface markers analysis (Figure 2B). Remarkably, treatment of these β (CD11b, CD11c, CD13, CD33, CD36, and CD115), indi- primary cells with the proinflammatory cytokines IL-1 α cating that our CD34+ HPC culture protocol also gave and TNF- dramatically reduced the level of CXCL14 rise to monocytes (data not shown). Collectively, the mRNA, which is in striking contrast to inflammatory “true” CXCL14-responsive cells carry many features of chemokines, as exemplified here by the tremendous in- “early” monocytes, i.e., CD14+ cells that appear early duction of CCL20 mRNA (Moser et al., 2004; Schaerli and Moser, 2005). Primary keratinocytes ceased to pro- during in vitro differentiation of CD34+ HPCs. duce CXCL14 mRNA during prolonged monolayer cul- Next, we examined CXCL14-induced migration of tures (>4 passages); also, CXCL14 mRNA was not de- CD14+ cells from human peripheral blood (see Figure tected in immortal keratinocyte and fibroblast lines. S1 in the Supplemental Data available with this article Unfractionated tissue from small intestine served as online). Among the different subsets of monocytes positive control for both CXCL14 and CCL20. Thus, un- characterized by CD14 and CD16 expression, the one der physiological conditions, CXCL14 may be involved expressing highest levels of CD14 (CD14high) showed in homeostatic as opposed to inflammatory immune best CXCL14 responsiveness. DCs, either isolated from processes. blood or generated in vitro from monocytes, did not Chemokines are rapidly truncated and functionally respond. The CD14high blood monocytes resemble in + + modified by numerous proteases that represent an ad- vitro generated CD34 HPC-derived CD14 DC precur- ditional level of control in leukocyte traffic within tis- sors in terms of migration and phenotypic characteris- sues (Moser et al., 2004). CXCL14 is unique among all + tics and are referred to hereafter as CD14 DC precur- chemokines in its very short N terminus consisting of sors to underscore their differentiation potential (see two residues preceding the first Cys residue (NH2- below). SKCKC) (Hromas et al., 1999; Frederick et al., 2000; Cao et al., 2000). Therefore, it was important to confirm that Functional CXCL14 Is Present at Strategic Locations the keratinocyte-derived natural form of CXCL14 was in Normal Human Skin active and identical in structure to the synthetic form Tissue recruitment and localization of blood leukocytes used throughout our migration studies with CD14+ DC requires transendothelial migration followed by chemo- precursors. Natural CXCL14 was purified from superna- taxis, which are two distinct processes enabling the tants of primary keratinocyte cultures (<3 passages) colocalization of mobilized leukocytes with chemokine- and then subjected to mass spectroscopic and amino- producing tissue cells (Moser et al., 2004; Campbell et terminal sequence analyses (Figure 2C). The final frac- Immunity 334

Figure 2. Functional CXCL14 Is Present in Healthy Human Skin (A) Localization of CXCL14 protein in normal skin. Immunohistochemistry was performed on frozen tissue sections from healthy abdominal skin. CXCL14 protein (red staining) is detected in vascular/perivascular areas within the dermis and in the epidermis (left). Asterisk marks cut hair sheath and the inset depicts staining with a control Ab. Analysis of serial skin sections (middle and right) demonstrates overlapping expression of CXCL14 and CD31 (blood and lymphatic vessels; open arrowheads), but not podoplanin (lymphatic vessels; filled arrowheads) within the superficial dermal plexus. (B) Examination of CXCL14 and CCL20 mRNA in freshly isolated and cultured human skin cells. Northern blot analysis were performed with total RNA from keratinocytes (K) or adherent dermal cells (D), either freshly isolated or activated for 18 hr by 10 ng/ml IL-1β/TNF-α,from keratinocyte and fibroblast (F) lines in passage 3 or related immortalized lines (HaCat for K, MDX12 for F); RNA from normal small intestinal tissue served as positive control. Bottom panel shows ethidium bromide-stained RNA as loading control. (C) Primary keratinocytes produce CXCL14. The inset shows the elution profile of the reversed-phase C4 column; the indicated pooled fractions were then further purified over a CN-propyl column. CXCL14 protein was identified in the fraction eluting at 32.7 min. (D) Migration of CD14high monocytes to natural and synthetic CXCL14, and to a series of synthetic CXCL14 variants with amino-terminal extensions. Responses were evaluated in a modified Boyden chamber chemotaxis assay and were expressed as numbers of migrated cells present in five random high-power fields (5HPF); background migration was <15 cells/5HPF. Error bars show mean ± SEM. tion contained a protein with MW = 9414 and the amino- sions were inactive, demonstrating that slight amino terminal sequence NH2-SKCKCSRKGP, indicating that acid sequence variations at the amino terminus had a keratinocyte-derived CXCL14 is identical to the syn- profound effect on CXCL14 activity. Finally, in cells re- thetic form. More importantly, natural CXCL14 attracted covered from healthy skin, CXCL14-responsive cells monocytes with equal or even slightly increased po- were identified as CD14+CD1a− mononuclear cells, tency than synthetic CXCL14 (Figure 2D). In contrast, whereas, due to elevated background migration, re- synthetic CXCL14 forms with amino-terminal exten- sponsiveness of the more numerous CD1a+CD14− cells Localization and Differentiation of Epidermal DCs 335

to CXCL14 could not be determined (see Figures S2 tures using submersed monolayers of primary keratino- and S3). Together, CXCL14 protein is produced at stra- cytes, suggesting that continuous keratinocyte differ- tegic locations for extravasation and epidermal local- entiation, a prerequisite for the formation of proper epi- ization of CXCL14-responsive cells. Accordingly, we dermal architecture, is required for DC development. propose that this chemokine controls the recruitment This situation resembles the discrepant CXCL14 ex- of CD14+ DC precursors, which may lead to their in pression in keratinocyte cultures and EEs (Figures 2B situ differentiation. and 3B), although we do not propose that CXCL14 is a DC differentiation factor. The importance of cellular Localization and Survival of CD14+ DC Precursors niches was further illustrated by the finding that dermal in Human Epidermal Equivalents fibroblasts did not support LC differentiation but rather The hypothesis that epidermal tissue has an influence promoted differentiation into (CD14+, CD206+) macro- on CD14+ DC precursors was tested in an artificial epi- phages and (CD1a+, DC-SIGN+, Factor XIIIa+) interstitial dermis model. Epidermal equivalents (EEs) were gener- DCs (see Table S1; Chomarat et al., 2000). In summary, ated from epidermal stem cells derived from outer root the epidermal environment contributes to three distinct sheaths (ORS) of healthy human hair follicles (Limat and steps that may affect in situ development of DCs, Hunziker, 2002). These EEs lack hematopoietic cells but namely the suprabasal positioning of CD14+ DC precur- express typical skin differentiation markers and show sors as well as their long-term survival and differenti- histological features of normal human epidermis, in- ation. cluding a basal layer of cuboidal keratinocytes, followed by multiple (4–8) layers of progressively flattened Epidermal Contact Turns CD14+ DC Precursors keratinocytes, a stratum granulosum (3–5 layers), and into Functional LC-like DCs a stratum corneum, which provides barrier function in To study the degree of CD14+ DC precursor differentia- skin (Figure 3A). EEs stained positive for CXCL14, fur- tion, hematopoietic cells were analyzed by flow cyto- ther underscoring tissue integrity (Figure 3B). First we metry after proteolytic digestion of immune-reconstitu- examined the effect of artificial epidermis on CD14+ DC ted EEs. After 10 days of epidermal coculture, a distinct precursor positioning and survival in “sandwich cultures” population of hematopoietic cells (CD45+) expressing made from fully developed EEs and CFSE-labeled high levels of MHC class-II molecules (HLA-DR) was CD14+ DC precursors. After 7 days of culture, all CFSE+ recovered showing cell-size properties (forward- and cells have entered the suprabasal layers in EEs (Figure side-scatter profiles) similar to those seen in in vitro 3C). The mechanism underlying intraepidermal posi- monocyte-derived DCs but clearly distinct from input tioning of CD14+ DC precursors is not clear at present. cells (Figure 4A). The large majority of CD45+HLA- Furthermore, the epidermal environment provided sur- DRhigh cells has lost the monocyte marker CD14 but vival signals, since counting of fluorescent cells within acquired the myeloid DC markers CD1a and CD1c (Fig- EEs revealed that ~1/3 of input CFSE+ cells had sur- ure 4B). A substantial fraction of cells expressed sur- vived (Figure 3D). Also, a similar fraction of cells was face Langerin, a C-type lectin associated with Birbeck recovered after culturing of CD14+ DC precursor cells granules (Valladeau et al., 2000), high-affinity IgE recep- on top of primary keratinocyte monolayers. This is in tors (FcIg␧-RIα), CD40, and DEC-205/CD205, indicating striking contrast to CD14+ DC precursors that rapidly the development of DCs with LC-like characteristics died during culture in the absence of keratinocytes or (Valladeau et al., 2000; Novak et al., 2003; Inaba et al., survival factors. Flow cytometry revealed that CFSE+ 1995). Other LC markers (e.g., E-Cadherin) could not be cells recovered from EEs did not proliferate (data not analyzed due to sensitivity to the proteolytic treatment shown). of EEs. By contrast, the few surviving cells recovered Next we examined if suprabasal positioning and sur- after culturing of CD14+ DC precursors for 10 days in vival was coupled with CD14+ DC precursor differentia- the absence of epidermis but in the presence of the tion. The inset of Figure 3C documents the dendritic same media supplements retained monocytic charac- morphology in most CFSE+ cells present within the teristics (data not shown). EEs. Also, numerous suprabasal hematopoietic (CD45+) Next we examined the effect of maturation stimuli on cells were detected in vertical sections of CD14+ DC CD14+-derived progenitor cells within EEs. For this, en- precursor cell-reconstituted EEs (Figure 3E). Some of tire immune-reconstituted EEs at 10 days of culture these cells expressed the DC markers CD1a and Lang- were transferred to culture dishes containing medium erin (see below) and showed DC-like morphology (Fig- supplemented with or without bacterial endotoxin (LPS) ure 3F). We found that CD14+ DC precursor differentia- and TNF-α, followed by incubation for another 2.5 days. tion was much more improved in EEs generated from HLA-DR and the costimulatory molecules CD80 and ORS cultures than from interfollicular keratinocytes. CD86 were already expressed on immature LC-like Similarly critical were the coculture conditions, includ- cells but were further upregulated during subsequent ing the composition of the growth factor supplement treatment with maturation stimuli, as evidenced by flow and the time point when CD14+ DC precursors were cytometry (compare MFI values in Figure 4C). Blood mixed with developing epidermis. Under these condi- CD14+ DC precursor cells, in contrast, lacked CD80 tions, immune-reconstituted EEs contained 3.2 ± 0.9 and expressed intermediate levels of CD86 and HLA- CD45+ cells/mm (4 independent experiments), which is DR (not shown). Interestingly, the DC maturation mark- similar to the number of LCs in the epidermis of normal ers CD83 and CCR7 were not detected on “mature” LC- human skin (Proksch et al., 1996). Importantly, develop- like cells, in agreement with epidermal production of ment of DC characteristics was not detected in cocul- TGF-β (Figure S4) and other potential inhibitors (Strobl Immunity 336

Figure 3. Localization and Survival of CD14+ DC Precursors in EEs (A) EEs generated from outer root sheath cells of hair follicles show well-developed epidermal architecture. Sections were paraffin embedded and stained with hematoxylin- eosin (HE). (B) EEs are highly positive for CXCL14 protein, as determined by immunohistochemistry in frozen tissue sections; negative control staining was performed with an isotype- matched control Ab. (C) CD14+ DC precursors move into suprabasal location during sandwich culture with fully developed EEs. CD14+ DC precursors were labeled with CFSE, which allowed tracking of cells by fluorescence microscopy. Enlargement of the top view (inset) reveals CFSE+ cells with dendritic morphology. (D) EEs and keratinocytes support survival of CD14+ DC precursors. Fully developed EEs were placed onto culture inserts seeded with CFSE-labeled CD14+ DC precursors (black bar) or, alternatively, CFSE-labeled CD14+ DC precursors were cultured on submersed monolayers of primary human keratinocytes (gray bar) and after 7 days CFSE+ cells were counted. As control (open bar), CD14+ DC precursors were cultured for 7 days in the absence of EEs or keratinocytes. Surviving cells are expressed as live (trypan blue- negative) cells in percent of number of input cells; data are representative of three independent experiments. Error bars show mean ± SEM. (E) Distribution of CD45+ cells after coculture of CD14+ DC precursors with developing EEs. Frozen sections of EEs were stained with an Ab to CD45. (F) Immune-reconstituted EEs as in (E) were stained with an Ab to CD1a (green) and analyzed by fluorescence microscopy; nuclei were revealed by DAPI staining (blue).

and Knapp, 1999). Sorted (CD45+HLA-DR+) cells from ings demonstrate the dynamic changes in these mem- LPS/TNF-α-treated EEs were then tested for their abil- brane lamellae (Movie S1), resembling interdigitating ity to induce cell proliferation in a primary mixed-leuko- DCs (Miller et al., 2004). Veiled cells were more numer- cyte reaction, using highly pure naive CD4+ T cells as ous among emigrant cells from LPS/TNF-α-treated EEs responder cells. As negative and positive control APCs, than from medium control EEs, in agreement with their freshly isolated CD14+ DC precursors and in vitro gen- enhanced state of maturation (see below). erated, mature monocyte-derived DCs from the same Immunocytochemical analysis of combined emigrant donor were used. Of note, EE-derived cells were potent and EE-extracted cells confirmed the contrasting state stimulators of naive CD4+ T cells and prominent prolif- of maturation in cells derived from LPS/TNF-α-treated eration responses were already observed at an APC: EEs versus control EEs (Figure 5B). Treatment of imma- responder cell ratio of 1:1000 (Figure 4D). EE-derived ture DCs with inflammatory stimuli results in a burst of cells were less potent than control DCs, which may be MHC class II gene expression, which is followed by the explained by the harsh treatment employed for extract- relocation of new and preformed MHC class II mole- ing these APCs out of EEs. Freshly isolated CD14+ DC cules to the cell surface (Cella et al., 1997; Pierre et al., precursors failed to induce substantial T cell prolifera- 1997; Chow et al., 2002). In agreement, CD14+-derived tion responses. cells from LPS/TNF-α-treated EEs showed strong cell- The effect of EEs on DC morphology and marker dis- surface HLA-DR staining, whereas in cells from control tribution was examined by microscopy. Emigrant cells EEs, intracellular HLA-DR staining predominated (Fig- of immune-reconstituted EEs revealed a “veiled” mor- ure 5B, and below). EE-derived cells had long hair-like phology, which is typical for skin emigrant cells (Figure membrane processes, highlighted by the prominent 5A; Schuler et al., 1985; Larsen et al., 1990; Romani et HLA-DR staining in LPS/TNF-α-exposed cells. Further- al., 1989, Lenz et al., 1993). Time-lapse video record- more, 50% of CD14+-derived cells from LPS/TNF-α- Localization and Differentiation of Epidermal DCs 337

Figure 4. EEs Promote Differentiation of CD14+ DC Precursors into LC-like Cells (A) Marker expression and cell size of freshly purified CD14+ DC precursors that were rou- tinely used for immune reconstitution of EEs (top) and gating strategy to identify the progeny of CD14+ DC precursors in total cell sus- pensions of trypsinized immune-reconstituted EEs. Flow cytometry was performed on live (propidium-iodide-negative) cells. Note the increased FSC/SSC properties in cultured CD45+HLA-DR+ cells. (B) The progeny of CD14+ DC precursors express markers for DCs and LCs. After 10 days of coculture, CD45+HLA-DR+ cells were analyzed by flow cytometry for the markers as indicated. Numbers within individual plots indicate the percentage of marker-positive cells; data representative of three experiments are shown. (C) Immune-reconstituted EEs were cultured for another 2.5 days in the presence or absence of LPS and TNF-α, and recovered CD45+HLA-DR+ cells were analyzed by flow cytometry for expression of HLA-DR (circles), CD80 (squares), and CD86 (triangles). Filled and empty symbols refer to EEs cultured in the presence or absence of LPS and TNF-α. Data are representative of three independent experiments. (D) Mature LC-like cells had potent antigen- presentation and costimulation function. Live CD45+HLA-DR+ cells (filled circles) recovered from LPS/TNF-α-treated immune-reconstituted EEs, mature monocyte-derived DCs (filled triangles), and freshly isolated CD14+ DC precursors (open squares) were analyzed in mixed-leukocyte reactions with naive heterologeous CD4+ T cells as responder cells. The three types of APCs were derived from the same donor. Proliferation was measured by [3H]-thymidine incorporation, and the data are representative of two independent experiments. Error bars show mean ± SEM.

treated EEs uniformly expressed the maturation marker Langerin staining in LC-like cells from control EEs was DC-lysosome-associated membrane glycoprotein (DC- weak and predominantly intracellular, whereas treat- LAMP/CD208) (Saint-Vis et al., 1998), whereas the large ment with LPS plus TNF-α resulted in increased Lang- majority of cells from control EEs lacked this marker erin staining, part of which colocalized with cell-surface (Figure 5B, Table 1). Next, we examined the degree of HLA-DR. In summary, our epidermal coculture model LC differentiation by staining for Langerin (Valladeau et allowed efficient in situ differentiation of CD14+ DC pre- al., 2000). In agreement with the flow cytometry data cursors into LC-like cells with potent APC function. (Figure 4B), Langerin+ cells were readily detected in cells from control EEs; however, this staining was weak Discussion and mostly intracellular (Figure 5B). LPS/TNF-α treatment resulted in marked upregulation of Langerin, The molecular mechanisms underlying DC relocation to which has also been observed with in vitro generated inflammatory sites and tissue-draining LNs are becom- LCs (Valladeau et al., 1999, 2000; Geissmann et al., ing increasingly clear (Sallusto and Lanzavecchia, 2000). 2002). The frequency of Langerin+ cells among CD14+- The opposite is true for the traffic control of DC precur- derived cells from LPS/TNF-α or medium-treated EEs sors under steady-state conditions, notably in the set- compared well with HLA-DR+ emigrant cells from epi- ting of DC development in epithelial tissues. CCL2 was dermal sheets of normal skin (Table 1). proposed to control DC (and macrophage) develop- Confocal fluorescence microscopy confirmed the in- ment at inflammatory sites by means of attracting tracellular-to-cell surface redistribution of HLA-DR dur- CCR2+ blood monocytes (Lu et al., 1998; Merad et al., ing in situ exposure of LC-like cells to LPS and TNF-α 2002). However, this inflammatory chemokine is un- (Figure 6A; Cella et al., 1997; Pierre et al., 1997; Chow likely to play a role in DC development under steady- et al., 2002). The distribution of HLA-DR in LC-like cells state conditions. CCL20, the single ligand for CCR6, from control EEs largely colocalized with the late endo- attracts skin-emigrant CD14+ cells as well as LC-like somal and lysosomal marker LAMP-1/CD107a; how- cells derived from CD34+ HPCs or monocytes (Larre- ever, during the treatment with LPS/TNF-α, the LAMP-1 gina et al., 2001; Dieu et al., 1998; Yang et al., 1999; staining clustered intracellularly, whereas HLA-DR Sato et al., 2000). In CCR6-deficient mice, DCs are ab- staining gained in intensity and moved to cell surface. sent in the subepithelial dome of Peyer’s patches within This situation is further documented in video recordings intestinal mucosa, whereas DCs in normal skin are not of sequential confocal planes (Movies S2–S5). Finally, affected (Cook et al., 2000; Varona et al., 2001). Also, Immunity 338

Figure 5. Morphologic and Phenotypic Char- acteristics of Immature and In Situ Matured LC-like Cells (A) Phase-contrast microscopy of live emigrant cells collected after 2.5 days treatment of immune-reconstituted EEs with LPS and TNF-α. Note numerous veiled-type membrane protrusions that are continuously re- tracted and newly formed (Movie S1). Scale bars equal 10 ␮m. (B) Immunocytochemical analysis of cytospin preparations with cells recovered from immune-reconstituted EEs after treatment for 2.5 days with (LPS/TNF) or without (Me- dium) LPS and TNF-α. Cells analyzed for indicated markers included a combination of emigrant and EE-extracted cells. Scale bars equal 10 ␮m; images are representative of three independent experiments.

CCL20 is low in normal human skin but is highly upreg- under steady-state conditions. First, CXCL14 is readily ulated in inflammation, which is in line with the pres- detected in healthy human skin and is not further ence of CCR6 on effector/memory T cells and a role for upregulated in skin diseases, such as psoriasis and this chemokine system in inflammatory as opposed to atopic dermatitis (data not shown), indicating a homeo- homeostatic processes (Moser et al., 2004). static function for this ill-defined chemokine. Second, Here, we provide evidence for our hypothesis that CXCL14 protein is present on blood vessels in the su- CXCL14 contributes to the development of LC-like cells perficial dermal plexus as well as within the epidermis,

Table 1. Immunocytochemical Analysis of Epidermis-Derived LC-like Cells

Immunolabeled Cells Tissue Treatment HLA-DR DC-LAMP Langerin EEa Medium 10.5% (8.2%–12.8%)b 0.1% (0.0%–0.1%) 4.2% (1.5%–7.0%) LPS/TNF 10.3% (7.1%–13.6%) 4.9% (4.7%–5.2%) 4.1% (3.1%–5.0%) Epidermisc Medium 17.0% (13.5%–20.5%) 12.7% (10.7%–14.7%) 7.3% (4.8%–11.4%) a Immune-reconstituted EEs were treated with (LPS/TNF) or without (Medium) LPS and TNF-α for 2.5 days, and cytospins of Ficoll Hypaque- enriched cell preparations containing emigrated cells together with EE-extracted cells were analyzed by immunocytochemistry for marker expression. b Number of marker-positive cells in percent of total cells that included leukocytes and an excess of keratinocytes. The fraction of HLA-DR+ cells serves as an internal reference value for evaluating the degree of DC-LAMP and Langerin expression. Total cells counted ranged from 1020 to 6240, and numbers in parentheses indicate the range of marker-positive cells between cytospin preparations of two independent experiments. c For comparison, epidermal sheets from normal human skin were cultured in medium for 2.5 days, and emigrant cells were analyzed by immunocytochemistry. Note that this procedure resulted in the release of numerous tissue cells. Localization and Differentiation of Epidermal DCs 339

Figure 6. Mobilization and Intracellular Distri- bution of DC Markers during In Situ Matura- tion of LC-like Cells Confocal immunofluorescence microscopy of single cells within cytospin preparations as described in Figure 5B. LPS/TNF and Me- dium refer to the treatment of immune-reconstituted EEs with or without LPS and TNF-α. Scale bars equal 10 ␮m, DIC stands for digi- tal interference contrast images, and Overlay refers to the combined fluorescence signals in single midplane sections. Asterisk de- notes two unlabeled keratinocytes. Data from one out of three independent analyses are shown.

two distinct sites defining leukocyte extravasation and quired a LC phenotype, characterized by expression of tissue localization, respectively (Moser et al., 2004). DC and LC markers (Banchereau et al., 2000; Shortman Third, CXCL14 is uniquely selective for CD14+ DC pre- and Liu, 2002; Steinman et al., 2003). We wish to point cursors, either generated in vitro from CD34+ HPCs or out that CD14+ DC precursor development in our model isolated as CD14high monocytes from blood and using EEs did not depend on exogenous DC survival CD14+CD1a− cells from dermal skin tissue, respec- and differentiation factors. In fact, we have no knowl- tively. This chemokine does not attract other types of edge of an alternative system yielding a similar degree myeloid cells (including blood DC subsets and mono- of DC differentiation in the absence of supplements cyte-derived DCs/LCs) or other cell lineages (including containing combinations of GM-CSF, Flt-3L, TNF-α, T, B, and NK cells). This restricted target cell selectivity TGF-β, etc. In addition, these LC-like cells matured in is in clear contrast to other chemokines, which gen- situ in response to inflammatory stimuli, as assessed erally attract multiple types of leukocytes. Of note, the by HLA-DR and DC-LAMP expression as well as acquisi- minor subset of CD14lowCD16+ blood monocytes showed tion of veiled cell morphology (Schuler et al., 1985; only limited chemotaxis toward CXCL14. It was demon- Larsen et al., 1990; Romani et al., 1989; Valladeau et strated that this type of monocytes or corresponding al., 1999, 2000; Geissmann et al., 2002). Finally, in situ phagocytic cells preferentially undergoes differentiation matured LC-like cells were functional, as evidenced by into DCs during reverse transmigration, which occurs potent induction of proliferation responses in naive T when tissue-resident phagocytes enter the lymphatic cells. system (Randolph et al., 1998, 2002). In summary, our model assigns a critical function to Abundant expression of CXCL14 in the epidermis led CXCL14 and epidermal tissue in the homeostasis of hu- us to propose that CXCL14 mediates the epidermal re- man skin DCs. CXCL14 may control the epidermal recruitment of CXCL14-responsive CD14+ DC precursors, cruitment of circulating DC precursors, whereas intact thereby promoting their in situ differentiation into func- epidermal tissue provides structural and functional tional DCs. In a coculture model employing artificial niches for DC development, i.e., CXCL14 and epidermal EEs, we show that intraepidermal localization had pro- tissue work in sequential but independent fashion. Hu- found effects on CD14+ DC precursors. CD14+ DC pre- man CXCL14 mRNA is also expressed in other epithe- cursors migrated into the suprabasal layer of keratino- lial tissues, including intestinal tract, airways, and kid- cytes, which is the primary location of epidermal LCs. ney (Kurth et al., 2001; Hromas et al., 1999; Frederick EEs also provided survival signals that prevented et al., 2000), suggesting that our model of DC homeo- extensive cell death of CD14+ DC precursors. Most im- stasis may be extended to skin-unrelated tissues. The portantly, a large fraction of CD14+ DC precursors ac- present coculture system using immune-reconstituted Immunity 340

EEs will provide an invaluable tool for future studies on for 6–48 hr followed by a Ficoll purification of cells present within LC differentiation. the culture medium.

Experimental Procedures Epidermal Equivalents and Cocultures Epidermal equivalents (EEs) were generated from ORS cells as de- The data on antibodies and cytokines, CXCL14 receptor stainings, scribed (Limat and Hunziker, 2002). In brief, 200,000 epidermal and purification of natural CXCL14 are described in Supplemental stem cells, derived from a 7–14 day preculture of ORS of hair folli- Experimental Procedures. cles, were seeded in 500 ␮l of K medium into a cell culture insert (Corning Costar) carrying 200,000 irradiated (70 Gy) primary dermal Culture Media fibroblasts on the undersurface, i.e., stem cells were separated RPMI medium consisted of RPMI 1640 plus 2 mM L-glutamine, 1% from fibroblasts by the membrane insert. Dermal fibroblasts were nonessential amino acids, 1% sodium pyruvate, 50 ␮g/ml penicil- derived from primary cultures (p < 5). “Sandwich culture” refers to lin/streptomycin, 5 × 10−5 M β2-ME (all Invitrogen, Basel, Switzer- intact EEs that were placed onto the membrane of new inserts, land), and 10% FCS (Biological Ind., Israel). DMEM medium con- containing fibroblasts on the lower side and DC precursors on the sisted of DMEM (4.5 mg/ml glucose; Invitrogen) plus 10% FCS, 2 upper side. DC precursors were seeded into the insert and allowed mM L-glutamine, 1% nonessential amino acids, 50 ␮g/ml penicillin/ to attach on the membrane for 1 hr before medium was completely streptomycin (Invitrogen), 5 × 10−5 M β2-ME. F medium consisted removed and cells were covered with the EEs. For generation of of 3 parts DMEM and 1 part Ham’s F12 (PAA Labs, Austria) plus 20 immune-reconstituted EEs, inserts were cultured in K+ medium un- mM HEPES, 0.135 mM adenine (Sigma, St. Louis, MO), 5 ␮g/ml til stem cells reached confluence, then washed several times with insulin (Invitrogen), 10 ng/ml EGF, 0.4 ␮g/ml hydrocortisone (Sigma), RPMI and covered with 200,000 freshly isolated autologous high 2 nM triiodthyronine (Sigma), 100 U/ml Penicillin, 100 ␮g/ml strepto- CD14 monocytes for 2 hr in RPMI/F12 medium. Then, all me- mycin, 10% FCS. K medium was F medium plus 10 ng/ml cholera dium in the upper well was carefully removed and 1 ml RPMI/F12 toxin (Sigma), and K+ medium was K medium plus 50 ␮g/ml medium in the lower well was replaced every day until analysis L-Ascorbic acid (Sigma). RPMI/F12 medium consisted of 3 parts (after 1–2 weeks). For in situ maturation, immune-reconstituted EEs RPMI medium and 1 part Ham’s F12 plus 20 ng/ml EGF, 5 mM were transferred using forceps to new tissue culture dishes con- HEPES and 3.5 mg/ml D-glucose. taining culture medium with or without 500 ng/ml LPS and 10 ng/ ml TNF-α, and the EE were cultured for another 2.5 days before Blood Cell Isolation and Culture processing. Leukocytes were recovered by cutting the EEs into PBMCs were isolated from fresh blood of healthy individuals by small pieces and processing by digestion in 1× trypsin/EDTA for Ficoll centrifugation. HPCs were isolated from PBMCs by positive 30 min at 37°C, mechanical dispersion by vigorous pipetting, and ␮ selection of CD34+ cells using the MACS system (Miltenyi Biotech). filtering trough a 70 m pore mesh. Flow cytometry was done using − + + CD34+ cells were seeded at 35,000 cells/cm2/ml in DMEM medium bulk cells and gating on live (propidium-iodide ) CD45 HLA-DR plus 100 ng/ml GM-CSF, 2.5 ng/ml TNF-α, 10 U/ml SCF, 50 ng/ml cells, whereas cells were further purified by Ficoll centrifugation for Flt-3L. After 4 days, 0.5 ml/cm2 of fresh DMEM medium plus GM- preparation of cytospins. To track leukocytes within EEs, mono- ␮ CSF/TNF-α was added. Total monocytes for transwell assays were cytes were labeled with 0.5–5 M CFSE (Molecular Probes, Leiden, isolated from PBMCs by negative depletion of CD3+, CD8+, CD19+, The Netherlands) in PBS containing 5% FCS for 4 min at room tem- CD20+, CD34+, and CD56+ cells. CD14high monocytes for cocul- perature. tures were isolated from CD1c-depleted PBMCs by positive selection of CD14high cells. MDCs were isolated from lin-depleted Functional Assays PBMCs by positive selection of CD1c+ cells. PDCs were isolated Transwell chemotaxis assays were performed with mixed cell pop- from lin- and CD11c-depleted PBMCs and identified as CD4high ulations for 1.5 hr using 5 ␮m pore polycarbonate filter inserts CD123high cells in transwell assays. Monocyte-derived DCs and (Corning Costar Corp., Cambridge, MA). 48-well chemotaxis cham- LCs are described elsewhere (Sallusto and Lanzavecchia, 1994; bers of 5 ␮m pore size were used for the comparative analysis of Geissmann et al., 1998). DCs/LCs were matured in the presence of natural and synthetic CXCL14 preparations (Schaerli et al., 2004). 100 ng/ml LPS (Salmonella abortus equi) and used within 8–48 hr In the T cell proliferation assay, titrated numbers of 2× sorted for transwell assays. For functional studies, DCs were matured with CD45+CD33+ APCs (either progeny cells of cocultured CD14+ DC 500 ng/ml LPS and 10 ng/ml of each IL-1β and TNF-α for 2 days. precursors or in vitro generated, mature monocyte-derived DCs) Naive CD4 T cells were isolated from PBMCs by negative depletion were incubated with 50,000 heterologous naive CD4+ T cells in 250 of CD1c+, CD8+, CD14+, CD16+, CD19+, CD20+, CD25+, CD33+, ␮l RPMI medium per 96 U bottom well. [3H] thymidine incorporation CD34+, CD45RO+, CD56+, and γδ-TCR+ cells using FITC or PE- was measured after 5 days. Northern blot analyses with RNA ex- labeled secondary MACS Ab (Miltenyi Biotech). tracted by the RNAzole B method (Tel-Test Inc., Friendswood, TX) were performed as described elsewhere (Kurth et al., 2001). Skin Cell Isolation and Culture All skin cells were isolated from normal abdominal split skin (thick- Immunohistochemistry ness 0.5 mm). Dermal cells were isolated after digestion of split Normal human skin samples from abdominal and mammary reduc- skin with 1.25 U/ml Dispase II for 15 min (Roche Diagnostics tion surgery were frozen at −80°C in Tissue Tek O.C.T. Compound GmbH, Mannheim, Germany) to remove epidermis, and then re- (Sakura, Zoeterwoude, Netherlands). Freshly prepared 5 ␮m tissue maining dermal tissue was digested with 0.2 U/ml Collagenase D sections were blocked with human IgG at 3 mg/ml (Redimune, ZLB (Roche) for 45 min at 37°C in RPMI 1640 in a slowly tilting dish. Bioplasma AG, Bern), 10% donkey (Jackson ImmunoReasearch) or Adherent dermal cells were collected after plastic adherence for 2 rabbit (DakoCytomation) serum, 0.01% Tween 20, and 0.01% so- hr in complete RPMI or after stimulation with IL-1β/TNF-α (each dium azide in Tris-buffered saline. Acetone-fixed sections were in- 10 ng/ml) for 18 hr. Primary fibroblast lines were established from cubated overnight at 4°C with primary mouse Ab to CXCL14, CD31, adherent cells in F medium. Immortalized dermal fibroblast line CD1a, CD45, rabbit polyclonal Ab to podoplanin, murine isotype MDX12 was from MODEX Therapeutiques (Lausanne, Switzerland). control Ab IgG2a, IgG1, or rabbit IgG. Primary Ab were detected by Keratinocytes were derived from epidermal sheets peeled off from biotinylated donkey anti-mouse or donkey anti-rabbit Ab. Subse- split skin after digestion with Dispase II for 20 min at 37°C. Then, quent detection was performed by the ABC method (Kurth et al., epidermal sheets were trypsinized for 15 min at 37°C to obtain an 2001). EEs were either formaldehyde fixed and paraffin embedded epidermal cell fraction containing highly enriched keratinocytes. for classical haematoxylin-eosin stain or frozen in O.C.T. com- Primary keratinocyte lines were established in serum-free medium pound. Alternatively, formaldehyde- or acetone-fixed cytospins of (10744; Invitrogen) or in F medium by seeding fresh keratinocytes cells extracted from immune-reconstituted EEs (TNF/LPS- or me- in feeder dishes containing 4500/cm2 irradiated (70 Gy) dermal fi- dium-treated) and human epidermal cells (spontaneously emi- broblasts (<5 passages). Skin or epidermal emigrant cells were ob- grated) were labeled with primary HLA-DR, DC-LAMP, Langerin, tained by placing split skin or epidermal sheets on RPMI medium LAMP-1, and isotype control Ab in the presence of 1% saponin for Localization and Differentiation of Epidermal DCs 341

cell permeabilization. For confocal immunofluorescence micro- distinct chemokines expressed in different anatomic sites. J. Exp. scopy, cytospins were double labeled with Cychrome-conjugated Med. 188, 373–386. HLA-DR and either PE-conjugated Langerin or LAMP-1 Ab under Ebert, L.M., Schaerli, P., and Moser, B. (2005). Chemokine-medi- permeabilizing conditions. Cytospins were mounted in Mowiol and ated control of T cell traffic in lymphoid and peripheral tissues. Mol. examined using a Zeiss LSM510Meta confocal laser scanning Immunol. 42, 799–809. microscope. Frederick, M.J., Henderson, Y., Xu, X., Deavers, M.T., Sahin, A.A., Wu, H., Lewis, D.E., El Naggar, A.K., and Clayman, G.L. (2000). In Supplemental Data vivo expression of the novel CXC chemokine BRAK in normal and Supplemental data include three figures, one table, five movies, cancerous human tissue. Am. J. Pathol. 156, 1937–1950. and Supplemental Experimental Procedures and can be found with Geissmann, F., Prost, C., Monnet, J.P., Dy, M., Brousse, N., and this article online at http://www.immunity.com/cgi/content/full/23/ Hermine, O. (1998). Transforming growth factor beta1, in the pres- 3/331/DC1/. ence of granulocyte/macrophage colony-stimulating factor and interleukin 4, induces differentiation of human peripheral blood Acknowledgments monocytes into dendritic Langerhans cells. J. Exp. Med. 187, 961–966. We thank Drs. A. Banic, M. Konstantinescu, D. Erni, F. Fischer, I. Geissmann, F., Dieu-Nosjean, M.C., Dezutter, C., Valladeau, J., Harder, F. Russ, and R. Gmür (Dept. of Plastic and Reparation Sur- Kayal, S., Leborgne, M., Brousse, N., Saeland, S., and Davoust, J. gery, University of Bern) for skin tissue. We also acknowledge the (2002). Accumulation of immature Langerhans cells in human advice and technical support provided by Dr. T. Hunziker (Institute lymph nodes draining chronically inflamed skin. J. Exp. Med. 196, of Dermatology, University of Bern), Dr. A. Limat, and Regula 417–430. Stuber-Roos. This work was supported by grant 31-106583 from the Swiss National Science Foundation and grant 03.0441-2 from Greaves, D.R., Wang, W., Dairaghi, D.J., Dieu, M.C., De Saint-Vis, the Staatssekretariat für Bildung und Forschung. P.S. was sup- B., Franz-Bacon, K., Rossi, D., Caux, C., McClanahan, T., Gordon, ported by the Schweizerische Stiftung für Medizinisch-Biolog- S., et al. (1997). CCR6, a CC chemokine receptor that interacts with α ische Stipendien. macrophage inflammatory protein 3 and is highly expressed in human dendritic cells. J. Exp. Med. 186, 837–844. Hromas, R., Broxmeyer, H.E., Kim, C., Nakshatri, H., Christo- Received: December 6, 2004 pherson, K., II, Azam, M., and Hou, Y.H. (1999). Cloning of BRAK, a Revised: August 24, 2005 novel divergent CXC chemokine preferentially expressed in normal Accepted: August 25, 2005 versus malignant cells. Biochem. Biophys. Res. Commun. 255, Published: September 20, 2005 703–706. Inaba, K., Swiggard, W.J., Inaba, M., Meltzer, J., Mirza, A., Sasa- References gawa, T., Nussenzweig, M.C., and Steinman, R.M. (1995). Tissue distribution of the DEC-205 protein that is detected by the mo- Ardavin, C., Martinez del Hoyo, G., Martin, P., Anjuere, F., Arias, noclonal antibody NLDC-145. I. Expression on dendritic cells and C.F., Marin, A.R., Ruiz, S., Parrillas, V., and Hernandez, H. (2001). other subsets of mouse leukocytes. Cell. Immunol. 163, 148–156. Origin and differentiation of dendritic cells. Trends Immunol. 22, Jaksits, S., Kriehuber, E., Charbonnier, A.S., Rappersberger, K., 691–700. Stingl, G., and Maurer, D. (1999). CD34+ cell-derived CD14+ precur- Banchereau, J., Briere, F., Caux, C., Davoust, J., Lebecque, S., Liu, sor cells develop into Langerhans cells in a TGF-beta 1-dependent Y.J., Pulendran, B., and Palucka, K. (2000). Immunobiology of den- manner. J. Immunol. 163, 4869–4877. dritic cells. Annu. Rev. Immunol. 18, 767–811. Kurth, I., Willimann, K., Schaerli, P., Hunziker, T., Clark-Lewis, I., Campbell, D.J., Kim, C.H., and Butcher, E.C. (2003). Chemokines in and Moser, B. (2001). Monocyte selectivity and tissue localization the systemic organization of immunity. Immunol. Rev. 195, 58–71. suggests a role for breast and kidney-expressed chemokine Cao, X., Zhang, W., Wan, T., He, L., Chen, T., Yuan, Z., Ma, S., Yu, (BRAK) in macrophage development. J. Exp. Med. 194, 855–861. Y., and Chen, G. (2000). Molecular cloning and characterization of a Larregina, A.T., Morelli, A.E., Spencer, L.A., Logar, A.J., Watkins, novel CXC chemokine macrophage inflammatory protein-2 gamma S.C., Thomson, A.W., and Falo, L.D., Jr. (2001). Dermal-resident chemoattractant for human neutrophils and dendritic cells. J. Im- CD14+ cells differentiate into Langerhans cells. Nat. Immunol. 2, munol. 165, 2588–2595. 1151–1158. Caux, C., Vanbervliet, B., Massacrier, C., Dezutter-Dambuyant, C., Larsen, C.P., Steinman, R.M., Witmer-Pack, M., Hankins, D.F., Mor- Saint-Vis, B., Jacquet, C., Yoneda, K., Imamura, S., Schmitt, D., ris, P.J., and Austyn, J.M. (1990). Migration and maturation of Lang- and Banchereau, J. (1996). CD34+ hematopoietic progenitors from erhans cells in skin transplants and explants. J. Exp. Med. 172, human cord blood differentiate along two independent dendritic 1483–1493. cell pathways in response to GM-CSF+TNF alpha. J. Exp. Med. Lenz, A., Heine, M., Schuler, G., and Romani, N. (1993). Human and 184, 695–706. murine dermis contain dendritic cells. Isolation by means of a novel Cella, M., Engering, A., Pinet, V., Pieters, J., and Lanzavecchia, A. method and phenotypical and functional characterization. J. Clin. (1997). Inflammatory stimuli induce accumulation of MHC class II Invest. 92, 2587–2596. complexes on dendritic cells. Nature 388, 782–787. Limat, A., and Hunziker, T. (2002). Use of epidermal equivalents Chomarat, P., Banchereau, J., Davoust, J., and Palucka, A.K. (2000). generated from follicular outer root sheath cells in vitro and for IL-6 switches the differentiation of monocytes from dendritic cells autologous grafting of chronic wounds. Cells Tissues Organs 172, to macrophages. Nat. Immunol. 1, 510–514. 79–85. Chow, A., Toomre, D., Garrett, W., and Mellman, I. (2002). Dendritic Lu, B., Rutledge, B.J., Gu, L., Fiorillo, J., Lukacs, N.W., Kunkel, S.L., cell maturation triggers retrograde MHC class II transport from ly- North, R., Gerard, C., and Rollins, B.J. (1998). Abnormalities in mo- sosomes to the plasma membrane. Nature 418, 988–994. nocyte recruitment and cytokine expression in monocyte che- Cook, D.N., Prosser, D.M., Forster, R., Zhang, J., Kuklin, N.A., Ab- moattractant protein 1-deficient mice. J. Exp. Med. 187, 601–608. bondanzo, S.J., Niu, X.D., Chen, S.C., Manfra, D.J., Wiekowski, MacDonald, K.P., Munster, D.J., Clark, G.J., Dzionek, A., Schmitz, M.T., et al. (2000). CCR6 mediates dendritic cell localization, lym- J., and Hart, D.N. (2002). Characterization of human blood dendritic phocyte homeostasis, and immune responses in mucosal tissue. cell subsets. Blood 100, 4512–4520. Immunity 12, 495–503. Merad, M., Manz, M.G., Karsunky, H., Wagers, A., Peters, W., Dieu, M.C., Vanbervliet, B., Vicari, A., Bridon, J.M., Oldham, E., Aït- Charo, I., Weissman, I.L., Cyster, J.G., and Engleman, E.G. (2002). Yahia, S., Brière, F., Zlotnik, A., Lebecque, S., and Caux, C. (1998). Langerhans cells renew in the skin throughout life under steady- Selective recruitment of immature and mature dendritic cells by state conditions. Nat. Immunol. 3, 1135–1141. Immunity 342

Miller, M.J., Hejazi, A.S., Wei, S.H., Cahalan, M.D., and Parker, I. Elbe, A., Maurer, D., and Stingl, G. (1996). Generation of human (2004). T cell repertoire scanning is promoted by dynamic dendritic dendritic cells Langerhans cells from circulating CD34+ hematopoi- cell behavior and random T cell motility in the lymph node. Proc. etic progenitor cells. Blood 87, 1292–1302. Natl. Acad. Sci. USA 101, 998–1003. Valladeau, J., Duvert-Frances, V., Pin, J.J., Dezutter-Dambuyant, C., Moser, B., Wolf, M., Walz, A., and Loetscher, P. (2004). Chemokines: Vincent, C., Massacrier, C., Vincent, J., Yoneda, K., Banchereau, J., multiple levels of leukocyte migration control. Trends Immunol. 25, Caux, C., et al. (1999). The monoclonal antibody DCGM4 recog- 75–84. nizes Langerin, a protein specific of Langerhans cells, and is rapidly Novak, N., Kraft, S., and Bieber, T. (2003). Unraveling the mission internalized from the cell surface. Eur. J. Immunol. 29, 2695–2704. of FcepsilonRI on antigen-presenting cells. J. Allergy Clin. Immu- Valladeau, J., Ravel, O., Dezutter-Dambuyant, C., Moore, K., Kleij- nol. 111, 38–44. meer, M., Liu, Y., Duvert-Frances, V., Vincent, C., Schmitt, D., Da- Pierre, P., Turley, S.J., Gatti, E., Hull, M., Meltzer, J., Mirza, A., Inaba, voust, J., et al. (2000). Langerin, a novel C-type lectin specific to K., Steinman, R.M., and Mellman, I. (1997). Developmental regula- Langerhans cells, is an endocytic receptor that induces the formation of MHC class II transport in mouse dendritic cells. Nature 388, tion of Birbeck granules. Immunity 12, 71–81. 787–792. Varona, R., Villares, R., Carramolino, L., Goya, I., Zaballos, A., Gu- Proksch, E., Brasch, J., and Sterry, W. (1996). Integrity of the per- tierrez, J., Torres, M., Martinez, A., and Marquez, G. (2001). CCR6- meability barrier regulates epidermal Langerhans cell density. Br. J. deficient mice have impaired leukocyte homeostasis and altered Dermatol. 134, 630–638. contact hypersensitivity and delayed-type hypersensitivity responses. J. Clin. Invest. 107, R37–R45. Randolph, G.J., Beaulieu, S., Lebecque, S., Steinman, R.M., and Muller, W.A. (1998). Differentiation of monocytes into dendritic cells Yang, D., Howard, O.M.Z., Chen, Q.Q., and Oppenheim, J.J. (1999). in a model of transendothelial trafficking. Science 282, 480–483. Immature dendritic cells generated from monocytes in the presence of TGP-β1 express functional C–C chemokine receptor 6. J. Randolph, G.J., Inaba, K., Robbiani, D.F., Steinman, R.M., and Immunol. 163, 1737–1741. Muller, W.A. (1999). Differentiation of phagocytic monocytes into lymph node dendritic cells in vivo. Immunity 11, 753–761. Randolph, G.J., Sanchez-Schmitz, G., Liebman, R.M., and Schakel, K. (2002). The CD16(+) (FcgammaRIII(+)) subset of human monocytes preferentially becomes migratory dendritic cells in a model tissue setting. J. Exp. Med. 196, 517–527. Romani, N., Lenz, A., Glassel, H., Stossel, H., Stanzl, U., Majdic, O., Fritsch, P., and Schuler, G. (1989). Cultured human Langerhans cells resemble lymphoid dendritic cells in phenotype and function. J. Invest. Dermatol. 93, 600–609. Saint-Vis, B., Vincent, J., Vandenabeele, S., Vanbervliet, B., Pin, J.J., Ait-Yahia, S., Patel, S., Mattei, M.G., Banchereau, J., Zurawski, S., et al. (1998). A novel lysosome-associated membrane glycoprotein, DC-LAMP, induced upon DC maturation, is transiently expressed in MHC class II compartment. Immunity 9, 325–336. Sallusto, F., and Lanzavecchia, A. (1994). Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J. Exp. Med. 179, 1109–1118. Sallusto, F., and Lanzavecchia, A. (2000). Understanding dendritic cell and T-lymphocyte traffic through the analysis of chemokine receptor expression. Immunol. Rev. 177, 134–140. Sato, K., Kawasaki, H., Nagayama, H., Enomoto, M., Morimoto, C., Tadokoro, K., Juji, T., and Takahashi, T.A. (2000). TGF-beta 1 recip- rocally controls chemotaxis of human peripheral blood monocyte- derived dendritic cells via chemokine receptors. J. Immunol. 164, 2285–2295. Schaerli, P., and Moser, B. (2005). Chemokines: control of primary and memory T-cell traffic. Immunol. Res. 31, 57–74. Schaerli, P., Ebert, L., Willimann, K., Blaser, A., Roos, R.S., Loetscher, P., and Moser, B. (2004). A skin-selective homing mechanism for human immune surveillance T cells. J. Exp. Med. 199, 1265–1275. Schuler, G., Romani, N., and Steinman, R.M. (1985). A comparison of murine epidermal Langerhans cells with spleen dendritic cells. J. Invest. Dermatol. 85, 99s–106s. Shortman, K., and Liu, Y.J. (2002). Mouse and human dendritic cell subtypes. Nat. Rev. Immunol. 2, 151–161. Sleeman, M.A., Fraser, J.K., Murison, J.G., Kelly, S.L., Prestidge, R.L., Palmer, D.J., Watson, J.D., and Kumble, K.D. (2000). B cell- and monocyte-activating chemokine (BMAC), a novel non-ELR alpha- chemokine. Int. Immunol. 12, 677–689. Steinman, R.M., Hawiger, D., and Nussenzweig, M.C. (2003). Tolero- genic dendritic cells. Annu. Rev. Immunol. 21, 685–711. Strobl, H., and Knapp, W. (1999). TGF-beta1 regulation of dendritic cells. Microbes Infect. 1, 1283–1290. Strunk, D., Rappersberger, K., Egger, C., Strobl, H., Krömer, E.,