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Role of ChemR23 in directing the migration of myeloid and plasmacytoid dendritic cells to lymphoid organs and inflamed skin

Role of ChemR23 in directing the migration of myeloid and plasmacytoid dendritic cells to... BRIEF DEFINITIVE REPORT Role of ChemR23 in directing the migration of myeloid and plasmacytoid dendritic cells to lymphoid organs and inflamed skin 1 2 3 1 William Ver mi, Elena Riboldi, Valer ie Wittamer, Francesca Gentili, 4 1 4 5 Walter Luini, Simona Marrelli, Annunciata Vecchi, Jean-Denis Franssen, 3 2 4 4,6 David Communi, Luisa Massardi, Marina Sironi, Alberto Mantovani, 3 1 2,4 Marc Parmentier, Fabio Facchetti, and Silvano Sozzani 1 2 Department of Pathology and Section of General Pathology and Immunology, University of Brescia, 25123 Brescia, Italy Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Campus Erasme, B-1070 Brussels, Belgium Istituto Ricerche Farmacologiche “Mario Negri,” 20157 Milan, Italy Euroscreen S.A., B-6041 Gosselies, Belgium Centro IDET, Institute of General Pathology, University of Milan, 20133 Milan, Italy Chemerin is a chemotactic agent that was recently identified as the ligand of ChemR23, a serpentine receptor expressed by activated macrophages and monocyte-derived dendritic cells (DCs). This paper shows that blood plasmacytoid and myeloid DCs express functional ChemR23. Recombinant chemerin induced the transmigration of plasmacytoid and myeloid DCs across an endothelial cell monolayer. In secondary lymphoid organs (lymph nodes and tonsils), ChemR23 is expressed by CD123 plasmacytoid DCs and by CD1a DC-SIGN DCs in the interfollicular T cell area. ChemR23 DCs were also observed in dermis from normal skin, whereas Langerhans cells were negative. Chemerin expression was selectively detected on the luminal side of high endothelial venules in secondary lymphoid organs and in dermal endothelial vessels of lupus erythematosus skin lesions. Chemerin endothelial cells were surrounded by ChemR23 plasmacytoid DCs. Thus, ChemR23 is expressed and functional in plasmacytoid DCs, a property shared only by CXCR4 among chemotactic receptors. This finding, together with the selective expression of the cognate ligand on the luminal side of high endothelial venules and inflamed endothelium, suggests a key role of the ChemR23/ chemerin axis in directing plasmacytoid DC trafficking. DCs are potent antigen-presenting cells with a subpopulations can be recognized based on their CORRESPONDENCE Silvano Sozzani: unique ability to induce T and B cell responses morphology, location, and phenotypic profile. [email protected] as well as immune tolerance (1, 2). Among M-DC are located in peripheral nonlym- human peripheral blood DCs, at least two dis- phoid tissues at an immature functional state tinct subsets have been defined, based on the where they exert a sentinel function for incom- expression of CD11c, namely CD11c myeloid ing antigens. After antigen uptake, in the con- DCs (M-DC) and CD11c plasmacytoid DCs text of an inflammatory reaction, M-DC leave (P-DC; reference 1). In addition, a minor, still peripheral sites and reach secondary lymphoid poorly characterized population of CD11c organs by lymphatic vessels (5, 6). In contrast, BDCA-3 DCs has been described previously P-DC are typically absent in peripheral tissues (3). M-DC express myeloid markers such as under homeostatic conditions and are believed CD13 and CD33, the stimulatory receptor Ig- to leave the circulation to enter secondary lym- like transcript 1, and can produce high levels of phoid organs through the interaction with high IL-12 (4). Conversely, P-DC have a morphol- endothelial venules (HEVs; references 5, 6). ogy resembling plasma cells; are devoid of However, recently published papers demon- myeloid markers; express high levels of CD4, strated P-DC recruitment also in some inflamed CD62 ligand, and CD123; and can produce skin lesions, such as those associated with sys- high levels of IFN- (5–7). This dichotomy temic lupus erythematosus (LE; references 5, 8), observed in human blood is further extended at in nasal mucosa during allergic reactions, and in the tissue level, where P-DC and many M-DC tumors (for review see reference 5). JEM © The Rockefeller University Press $8.00 509 Vol. 201, No. 4, February 21, 2005 509–515 www.jem.org/cgi/doi/10.1084/jem.20041310 The Journal of Experimental Medicine Differences in distribution and tissue recruitment during inflammatory conditions between M-DC and P-DC may reflect distinct functional properties and be explained by spe- cific migratory mechanisms. Blood DC subsets express a similar pattern of chemotactic receptors, including CCR2, CCR5, CXCR2, CXCR3, CXCR4, and PAFR (9). How- ever, circulating P-DC, in contrast with M-DC, fail to mi- grate in response to inflammatory chemokines, whereas both subsets respond to lymph node–homing chemokines (i.e., CCL19 and CCL21) after maturation (9). Thus, the only chemotactic factor presently known for immature P-DC in vitro is CXCL12, the ligand of CXCR4. However, the mechanisms leading to the in vivo recruitment of P-DC to normal and inflamed tissues are still poorly understood. Chemerin is a novel protein identified as the natural ligand of ChemR23, a previously orphan protein G–coupled receptor expressed by immature DCs and macrophages (10). Chemerin was purified from ovarian cancer ascites and found to correspond to the product of the Tig-2 gene. Chemerin is expressed by many tissues, including spleen and lymph nodes, and is secreted as prochemerin, a poorly active pre- cursor protein. Extracellular proteases convert prochemerin into a full agonist of ChemR23 by proteolytic removal of the last six amino acids (10, 11). Previous work has shown that chemerin induces calcium fluxes, mitogen-activated protein Figure 1. Expression of ChemR23 in leukocytes. PBMCs were isolated kinase activation, and chemotaxis of monocyte-derived im- as described in Materials and methods and stained for ChemR23 expression. mature DCs, whereas LPS- or CD40L-mature DCs do not (a) PBMC subsets were analyzed by FACS analysis. One experiment repre- express functional ChemR23 receptors. The aim of this sentative of four independent experiments is shown. (b) PBMCs were ana- work was to investigate the functional expression of ChemR23 lyzed for the expression of CD14 and CD16 and three populations of mono- in human blood DC subsets. Here, we report that ChemR23 lo cytes were discriminated: CD14 CD16 , CD14 CD16 , and CD14 CD16 . is expressed and functional in M-DC and most importantly ChemR23 expression was investigated in monocyte subsets. The values in P-DC, a property shared only by CXCR4. The ChemR23 represent the mean  SD of positive cells or intensity of membrane stain- ligand, chemerin, is selectively expressed in HEVs and in LE ing (MFI; n  4; *, P  0.05; **, P  0.01 by one-way analysis of variance skin lesions, a prototypic inflammatory condition character- with Tukey post test). The flow cytometry histograms are the result of one experiment representative of four independent experiments. ized by tissue accumulation of P-DC. These data strongly suggest that the ChemR23/chemerin axis may play a key role in regulating the trafficking of P-DC. brane staining (mean fluorescence intensity [MFI]), with the predominant CD14 CD16 population having the lowest RESULTS AND DISCUSSION levels of membrane fluorescence (MFI). Expression and function of ChemR23 in blood The three major blood DC subsets were investigated on MHC class II leukocytes the basis of the expression of specific membrane markers: In a previous work, we reported that chemerin represents a BDCA-1 (M-DC), BDCA-4 (P-DC), and BDCA-3 (3). Fig. new chemotactic factor for monocyte-derived DCs (10). 2 a shows that ChemR23 was expressed by 40% of the cir- Experiments were performed with the aim to characterize culating M-DC population and by virtually all P-DC. DC the expression of ChemR23 in circulating leukocytes. Fig. 1 a maturation (85% CD83 cells), achieved in the presence of shows that ChemR23 expression was mainly present in LPS (M-DC) or influenza virus (P-DC), did not significantly MHC class II cells and in a minor subset of CD3 and affect the percentage of ChemR23 cells, and only induced a CD20 lymphocytes. Within blood mononuclear cells, decrease of the mean channel fluorescence (Fig. 2 a). On the MHC class II cells were the only cells that migrated in re- contrary, ChemR23 was only modestly expressed (30% pos- sponse to chemerin (unpublished data). The expression of itive cells) by BDCA3 DCs, regardless the state of maturation. ChemR23 was further investigated in MHC-II cells. Fig. 1 b Next, we evaluated the ability of chemerin, the ChemR23 shows that ChemR23 was expressed by the three major ligand, to induce DC transmigration across an endothelial cell monocyte subsets, namely CD14 CD16 , CD14 CD16 , monolayer. Chemerin induced a dose-dependent migration lo and CD14 CD16 . However, a statistically significant dif- of immature M-DC and P-DC, with a peak observed at 100 ference among the three populations was found both in pM. Compared with the migration observed in response to an terms of percentage of positive cells and intensity of mem- optimal concentration of CXCL12 (12 nM), chemerin in- 510 RECRUITMENT OF PLASMACYTOID DENDRITIC CELLS BY CHEMERIN | Vermi et al. BRIEF DEFINITIVE REPORT duced the migration of a similar percentage of M-DC, whereas P-DC migration was 50% of that induced by CXCL12. Therefore, chemerin is characterized by potency 100-fold higher (peak chemotactic concentration) and by a similar (M-DC) or slightly reduced (P-DC) efficacy (percent- age of migrated cells) as compared with CXCL12 (Fig. 2 b). Chemotaxis of both M-DC and P-DC to chemerin was com- pletely blocked in the presence of an anti-ChemR23 moAb, proving the involvement of ChemR23 in chemerin-induced DC migration (unpublished data). Of note, chemerin-induced migration of M-DC and P-DC was respectively 1.6- and 3.4- fold more efficient when assessed in transmigration assays than using bare filters. This result suggests that chemerin presenta- tion by endothelial cells is required for optimal interaction with ChemR23 DCs. It was reported previously that CXCR3 ligands increase P-DC migration in response to CXCL12 (12, 13). Therefore, P-DC migration was tested us- ing chemerin in combination with different concentrations of CXCL10 or CXCL12 in the lower wells. P-DC migration to chemerin was never increased by the presence of these two chemokines, indicating that ChemR23 does not cooperate with CXCR3 and CXCR4 for cell migration (unpublished data). Mature M-DC and P-DC did not migrate in response to chemerin (Fig. 2 b), but normally migrated in response to CCL19, one of the CCR7 ligands. It is noteworthy that ma- ture DCs retained high levels of ChemR23 expression, whereas they were unresponsive to the cognate agonist. Thus, the maturation process induces the uncoupling of ChemR23 with no major changes in its membrane expression. All in all, these results show that chemerin represents a new chemotac- tic factor for immature DCs. Most importantly, ChemR23 represents the only chemotactic receptor, in addition to Figure 2. Expression of functional ChemR23 in blood DC subsets. CXCR4, that is active in immature P-DC. Blood DC subsets were isolated as described in Materials and methods and Chemerin belongs to the cathelicidin/cystatin family of stained for ChemR23 expression. Maturation of DCs was achieved by incu- proteins, which includes precursors of bactericidal peptides bation with LPS (BDCA-1 ), influenza virus (BDCA-4 ), or LPS and influ- (cathelicidins), precursors of mediators active on leukocytes enza virus (BDCA-3 ) for 24 h. (a) DC subsets were analyzed by FACS analy- through protein G–coupled receptors (prokininogen, cathe- sis. (b) DC subsets were tested for their ability to migrate across an licidin precursors), as well as cysteine protease inhibitors endothelial cell monolayer in response to chemerin or to an optimal con- (cystatins). Elevated chemerin production was found in centration (12 nM) of CXCL12 (immature DCs) or CCL19 (mature DCs). The ovarian cancer ascites and in the synovial fluids of rheuma- values represent the mean  SD of triplicate data points and are repre- toid arthritis patients (10). Prochemerin transcripts were also sentative of at least five different experiments performed with indepen- dent donors. Note the different scale used in the left versus right panels. found in the skin of psoriatic subjects (14). In the bone stro- (iDC) Immature DCs. (mDC) Mature DCs. *, P  0.05 by paired Student’s t test. mal cell line ST2, the chemerin gene was reported to be in- duced by 1,25-dihydroxy-vitamin D3 (VitD3) and dexa- profiles. For instance, Langerhans cells express CD1a and Lan- methasone (15). Therefore, it is likely that chemerin represents gerin and are exclusively distributed within epithelial surface; an important signal involved in the recruitment of DCs dur- in contrast, the so-called “interstitial DCs,” typically repre- ing inflammation and autoimmune diseases, and at tumor sented by dermal DCs, are mainly CD1a and express DC- sites. The up-regulation of chemerin production by antiin- SIGN and the mannose receptor. In lymphoid organs, DC flammatory signals such as VitD3 and dexamethasone postu- subsets are recognizable in B and T cell compartments and in- lates a role for chemerin in all the conditions characterized clude, respectively, germinal center DCs and Langerhans- by a “type 2/alternative” state of activation, similar to that derived interdigitating DCs in T cell paracortical area (5, 6). present in tumor-associated leukocytes (16). The interfollicular area separates nodular lymphoid compart- ments. The latter is highlighted by the HEVs and populated Expression of ChemR23 by P-DC and M-DC in human tissues by many leukocyte populations, including lymphocytes, macro- At the tissue level, many DC subpopulations can be recog- phages, plasma cells, and at least two distinct DC subpopula- nized based on their morphology, location, and phenotypic tions, namely CD68 DC-SIGN DCs (17) and P-DC (5). JEM VOL. 201, February 21, 2005 511 The expression of ChemR23 was investigated by immu- nohistochemistry in lymph nodes and tonsils. In lymph nodes, numerous ChemR23 cells with a dendritic morphol- ogy were located in the interfollicular compartment (Fig. 3 a). These cells were identified by double immunofluorescence as DC-SIGN (Fig. 3 b) and CD1a cells (Fig. 3 c), two phe- notypic markers that identify distinct nodal myeloid DC pop- ulations (17). On the basis of their phenotype and location, these cells are believed to represent the nodal counterpart of peripheral blood DC-SIGN DCs (18, 19) that are recruited into the lymph nodes from the blood through the HEVs, or a population of dermal DCs migrated to the lymph node. ChemR23 reactivity was also present on CD123 and BDCA-2 P-DC (Fig. 3 d and its inset), either scattered as single cells or in clusters close to HEVs. ChemR23 expres- sion was not restricted to the interfollicular compartment and was also observed in sinus macrophages and in scattered cells within the germinal center (Fig. 3, a and d). This latter popu- lation coexpressed CD68 and represents germinal center macrophages (unpublished data). No reactivity was observed on B and T lymphocytes. These results indicate that ChemR23 expression is retained by P-DC and M-DC after their recruitment to secondary lymphoid tissues. The expression of ChemR23 was also investigated in Figure 3. ChemR23 expression in secondary lymphoid organs and normal skin. A strong reactivity for ChemR23 was identified normal skin. In secondary lymphoid organs, ChemR23 reactivity is ob- in cells located in the upper and intermediate dermis (Fig. 3 e). served in the interfollicular (IF) area and in tingible body macrophages of These ChemR23 cells displayed fusate/dendritic morphol- the germinal center (GC) (a and d). In the interfollicular areas, ChemR23 ogy and coexpressed DC-SIGN (Fig. 3 f) as well as the man- cells present a sparse perivenular distribution (a, black arrowheads), a DC nose receptor (Fig. 3 f, inset), thus corresponding to dermal morphology, and coexpress DC-SIGN (b, white arrowheads) and CD1a DCs (19). On the contrary, ChemR23 expression was never (c, white arrowheads). CD123 ChemR23 plasmacytoid DCs (P-DC; yellow detected in the cutaneous Langerhans cells (Fig. 3, e and f) cells) are also observed in the interfollicular area (IF) in proximity to a CD123 HEV (d), as cluster (d, white asterisk) or as sparse cells coexpressing and in the Langerhans cell–derived paracortical interdigitat- the P-DC–specific marker BDCA-2 (d, inset). In normal skin, ChemR23 ing dendritic cells that accumulate in the lymph node during reactivity is observed in dermal cells with fusate/dendritic morphology (e) dermatopatic lymphadenitis (reference 20 and not depicted). coexpressing DC-SIGN (f, white arrowheads) and mannose receptor (f, inset). These observations clearly indicate that chemerin is unlikely Indirect immunoperoxidase technique was applied (a and e); nuclei were to be a relevant chemotactic factor for the homing of counterstained with hematoxylin. Double immunofluorescence was per- Langerhans cell precursors to the skin, as well as for the mi- formed (b–d and f), using Texas red–conjugated secondary antibodies for gration of Langerhans cells to superficial lymph nodes. anti-ChemR23 (b–d, and e) and anti-BDCA-2 (d, inset) and FITC-conjugated secondary antibodies for anti–DC-SIGN (b and f), anti-CD1a (c), anti-CD123 Expression of chemerin by HEVs in secondary (d), anti-ChemR23 (d, inset), and antimannose receptor (MMR; f, inset). Magnification, 100 (a and d), 200 (e and f), and 400 (b, c, and d and f, insets). lymphoid organs Due to the abundance in ChemR23 cells in secondary lymphoid organs (lymph nodes and tonsils) and normal skin, in close proximity of HEVs that express chemerin suggests the distribution of chemerin was also investigated in these that chemerin represents an alternative/additional signal to tissues. Chemerin reactivity was primarily expressed in the CXCL12 for the recruitment of blood P-DC into lymph interfollicular area of lymph nodes and tonsils (Fig. 4 a), the nodes across HEVs (21). In this respect, it is intriguing that site where the majority of ChemR23 cells also localize. the pattern of chemerin immunoreactivity resembles that The strongest reactivity was observed in endothelial cells observed with an anti-CXCL12 antibody (reference 12 and with cuboidal/cylindrical morphology corresponding to HEVs. unpublished data). In addition to HEVs, chemerin expres- The identity of these chemerin cells was further confirmed sion was also found on sparse spindle cells located in the on the basis of the coexpression of Factor VIII, a specific en- perivenular area (Fig. 4 a). Based on their morphology, this dothelial cell marker (Fig. 4 b, inset), as well as CD123 (Fig. population is likely to represent stromal cells; their precise 4 a, inset) and CLA/Heca452 (Fig. 4 b), two markers also identity is currently under investigation. expressed by tissue P-DC (5). Of note, in HEVs, the highest HEVs were shown to produce CCL21, which accumu- expression of chemerin was observed at the luminal side lates at their luminal side. Other chemokines were detected on (Fig. 4 b). The presence of P-DC positive for ChemR23 the surface of HEVs, although they were produced by non- 512 RECRUITMENT OF PLASMACYTOID DENDRITIC CELLS BY CHEMERIN | Vermi et al. BRIEF DEFINITIVE REPORT HEV cells (e.g., CCL19, CXCL12, and CCL2; references 22, 23). In vitro experiments performed with five different types of endothelial cells of different origin (i.e., cord blood, dermis, iliac artery, and bone marrow) did not show any production of chemerin by endothelial cells both under basal conditions and after cytokine stimulation (TNF, IL-1, LPS, IFN, IFN, and combinations of these agonists; unpublished data). There- fore, it is unknown at the moment whether chemerin expres- sion is a prerogative of a specialized endothelium of an ana- tomical compartment, such as that of HEVs, or if chemerin is produced by stromal cells surrounding HEVs, and subse- quently transported through the fibroblastic reticular cell net- work to HEVs (for review see references 22, 23). Expression of chemerin by dermal endothelial cell of blood vessels in LE Nonresident DCs, including P-DC, accumulate in the skin in many inflammatory conditions (24). Thus, the expression of chemerin was assessed in tissue sections obtained from normal and inflamed skin. Chemerin expression was com- pletely absent in normal skin, including in endothelial cells of the papillary dermis vessels (Fig. 4 c). These data suggest that chemerin is unlikely to be responsible for skin recruit- ment of resident DCs in steady-state conditions. In recent Figure 4. Chemerin expression in secondary lymphoid organs, years, P-DC were directly implicated in the pathogenesis of normal skin, and cutaneous LE lesions. Immunostaining of lymph node LE (5, 8). In LE patients, the number of circulating P-DC is sections shows chemerin reactivity on endothelial cells and sparse periven- reduced, and these cells are present in the skin lesions, sug- ular spindle cells in the interfollicular area (a and b). Chemerin vessels gesting that P-DC are selectively recruited to the skin (8). correspond to HEV labeled by CD123 (a, inset), CLA (b), and Factor VIII (b, inset); Therefore, the expression of chemerin was examined in skin note the preferential expression of chemerin on the luminal side of HEV biopsies from nine cases of LE. Chemerin reactivity was ob- (b, arrowhead). Because CLA and CD123 also stain P-DC, these markers served in six out of the nine cases on the endothelial cells also identify P-DC surrounding HEV (a and b). Chemerin is absent in nor- mal skin (c). Chemerin cells are numerous in the dermis from a LE case (d). lining dermal vessels (Fig. 4, d–f). These endothelial cells did Most of these cells correspond to endothelial cells; in addition, scattered not express podoplanin (Fig. 4 g), a marker of lymphatic en- chemerin cells with fusate morphology are admixed to the lymphoid in- dothelium (25), indicating that they belong to blood vessels. filtrate (d and e). Rare chemerin cells are also noticeable in the epidermis In addition to endothelial cells, rare chemerin cells were (d). Double immunofluorescence demonstrated that, in LE lesions, CD123 also observed in the epidermis and in the dermis. In the epi- P-DC (e, inset, asterisk) are located around chemerin dermal vessels (e, inset; dermis, chemerin reactivity was present in scattered Langer- white arrowheads indicate the luminal side). On serial sections obtained hans cells (Fig. 4 d). In the dermis, sparse chemerin cells from a LE case, chemerin endothelial cells (f) did not coexpress the lym- were admixed to lymphoid infiltrates and displayed a fusate/ phatic endothelial cell marker podoplanin (g). Indirect immunoperoxidase dendritic morphology. These cells were found to corre- technique was applied (a and c–g); nuclei were counterstained with hema- toxylin. Double immunofluorescence was performed (a, inset; b; and e, in- spond to DC-SIGN dendrocytes (reference 26 and unpub- set) using Texas red–conjugated secondary antibodies for antichemerin lished data). Notably, in five out of six positive cases, P-DC (a and b, insets), anti-CLA (b), anti-CD123 (e, inset) and FITC-conjugated were present with variable density within the skin lesions secondary antibodies for anti-CD123 (a, inset), antichemerin (b and e, inset), and double immunofluorescence staining, demonstrating and anti–Factor VIII (b, inset). Magnification, 200 (b–d, f, g, and a and b, that CD123 P-DC are located in close proximity of insets); 400 (a); and 600 (e and its inset). chemerin dermal endothelial cells (Fig. 4 e, inset). This of P-DC across HEVs under steady-state conditions, and in suggests that chemerin local production can direct P-DC to the accumulation of P-DC observed in inflamed tissues. the skin in inflammatory conditions characterized by P-DC accumulation (5). MATERIAL AND METHODS In summary, this report shows that M-DC and P-DC Reagents. Recombinant chemerin was produced and purified as de- scribed previously (10). Human chemokines and cytokines used were: express ChemR23 in vitro and in vivo. Experiments per- CXCL12, CCL19 (PeproTech), IL-1 (Dompè), TNF (BASF Knoll), formed in vitro clearly show that chemerin represents a new IFN (Roussel Uclaf), and IFN (Roferon; Roche). RPMI 1640 was ob- chemotactic factor for blood M-DC and most importantly tained from GIBCO BRL. FCS was obtained from Hyclone. Cytokines for P-DC. The in vivo distribution of chemerin, located at and media were endotoxin free as assessed by the Limulus amoebocyte as- the luminal side of HEVs and inflamed blood dermal vessels, say (BioWhittaker Inc.). All reagents not otherwise specified were obtained strongly suggests that chemerin is involved in the migration from Sigma-Aldrich. JEM VOL. 201, February 21, 2005 513 Peripheral blood DC purification. PBMCs were isolated from buffy as specified before; human iliac artery ECs (27, 28) were grown in E199 with coats by Ficoll gradient (Amersham Biosciences) and peripheral blood 10% FCS, 10% human serum; human dermal microvascular ECs were ob- M-DC and P-DC were magnetically sorted with blood DC Ag (BDCA)-1 tained from PromoCell GmbH and cultured following the manufacturer’s and BDCA-4 cell isolation kits (Miltenyi Biotec), respectively, as described instructions; immortalized human dermal microvascular ECs (29) were previously (9). Blood M-DC and P-DC (10 cells/ml) were cultured in me- grown in E199 with 10% FCS, 10% human serum; human bone marrow mi- dium containing 1,000 U/ml recombinant human GM-CSF (Mielogen; crovascular ECs (30) were grown in E199 as HUVECs. Schering-Plough) and 10 ng/ml IL-13 (a gift from A. Minty, Sanofi Elf Bio Recherches, Labège, France) or 20 ng/ml IL-3 (ProSpec), respectively. Tissues and reagents. Surgical specimens from human tissues including Cells were matured by 24-h incubation with 100 ng/ml LPS (Escherichia coli normal skin (obtained from abdominal plastic surgery), skin biopsies from 055:B5; Sigma-Aldrich), or 20 ng of hemagglutinin/ml inactivated influ- nine cases of LE, reactive tonsils, and lymph nodes (removed for diagnostic enza virus strain A/Moscow/10/99 (a gift from T. De Magistris, Istituto purposes) were analyzed. Each specimen was snap frozen in isopentane pre- Superiore di Sanità, Rome, Italy). BDCA-3 MDC were isolated with cooled to liquid nitrogen temperature and stored at 80 C. Thin cryostat BDCA-3 cell isolation kit (Miltenyi Biotec), cultured in medium contain- sections were air dried overnight at room temperature and fixed in acetone ing 1,000 U/ml rhGM-CSF, 10 ng/ml IL-13, and 20 ng/ml IL-3 and ma- for 10 min before staining. Tissue expression of ChemR23 was evaluated tured by 24-h incubation with 100 ng/ml LPS and 20 ng of hemagglutinin/ by immunohistochemistry using two anti-ChemR23 monoclonal antibod- ml inactivated influenza virus (3). ies, namely 4C7 (IgG2b; dilution 1:15) and 1H2 (IgG2a; 1:15; reference 10). The indirect streptavidin–biotin complex immunoperoxidase tech- Flow cytometry. The expression of ChemR23 was analyzed by FACS us- nique was applied using a biotinylated anti-Ig multilinks secondary antibody ing two monoclonal antibodies (clone 4C7 and 1H2) described previously (1:20 dilution in Tris-HCl buffer, pH 7.4; Biogenex). The 4C7 and 1H2 (10). The expression of ChemR23 on PBMC subsets was analyzed by FACS moAbs properly reacted on frozen sections; no signal was observed in for- using: allophycocyanin-conjugated anti-CD3, PE-conjugated anti-CD20, malin-fixed tissue sections. Tissue expression of chemerin was detected us- and PE-conjugated anti-HLA-DR (Becton Dickinson); and allophycocyanin- ing two mouse monoclonal antibodies (clones 227C and 14G10; both conjugated anti-CD14 and PE-conjugated anti-CD16 (Exalpha). PE-conju- IgG1; dilution 1:30; 2-h incubation), directed against the peptide Cys- gated CD123 (IgG1; clone 9F5) and CD11c (IgG2b; clone S-HCL-3) were QRAGEDPHSFYFPGQFAFS, a common peptide for both active chemerin obtained from Becton Dickinson. Analysis of fluorescence was performed on and prochemerin. a FACStar Plus calibrated with Calibrite beads (Becton Dickinson). Double immunofluorescence stainings were performed using a set of primary antibodies, including CD123 (clone 7G3; mouse IgG2a; dilution Transendothelial migration assay. Transendothelial migration assay was 1:40; BD Biosciences), DC-SIGN (mouse IgG2b; dilution 1:5; BD Bio- evaluated using 24-well Costar Transwell chambers (5 m pore size; Corn- sciences), CLA (clone Heca452; rat IgM; dilution 1:100; BD Biosciences), ing). Human umbilical vein endothelial cells (HUVECs; passage 5) were CD1a (010, IgG1, dilution 1:30; Immunotech), Langerin (provided by subcultured to confluent monolayers on Transwell inserts and precoated with G. Trinchieri, Schering-Plough, Dardilly, France), and mannose receptor gelatin. Monolayers were rinsed with chemotaxis medium (RPMI 1640 con- (clone 3.29B1; mouse IgG1; dilution 1:50). Appropriate Texas red– and taining 10% FCS) before use. 100 l of DC suspension (1–2 10 cells/ml) FITC-conjugated isotype-specific secondary antibodies were used to reveal were seeded in the upper chamber and 600 l of chemoattractant or control the primary antibodies and sections were examined with a fluorescence mi- medium was added to the lower well. The chamber was incubated at 37 C croscope Olympus BX60, equipped with a Nikon digital camera. Anti– in humidified atmosphere in the presence of 5% CO for 4 h. Migrated cells human podoplanin Ab (clone 18H5, dilution 1:30; Abcam) recognizes lym- were recovered from the lower well and counted and the results were ex- phatic endothelium and was tested on frozen samples of breast carcinoma. pressed as the percentage of input cells. In some experiments, DC subsets This work was conducted in accordance with a protocol approved by the were cultured for 24 h in the presence or absence of a maturative stimulus Spedali Civili of Brescia Institutional Ethical Board and informed consent (LPS or influenza virus) and tested for their ability to migrate in response to was obtained from all patients. chemoattractants. PBMCs (10 10 cells/ml; 100 l of cell suspension per We thank M. Cella for the MR moAb. well) migration was tested in the same experimental conditions used for DCs. F. Gentili is supported by “Fondazione Beretta,” and E. Riboldi is supported by Migrated cells were collected and labeled for FACS analysis. “Centro di studio per il trattamento dello scompenso cardiaco.” We also thank the Associazione Italiana per la Ricerca sul Cancro, Ministero dell’Istruzione Università e Chemerin expression in endothelial cells. Chemerin expression was Ricerca, the Association for International Cancer Research (grant no. 04-223), and evaluated by RT-PCR. Total RNA samples (1 g) were reverse transcripted “Fondazione Berlucchi” for financial support to S. Sozzani. This work was also using the SuperScript II Rnase H Reverse Transcriptase (Invitrogen). PCR supported by the Belgian program on Interuniversity Poles of Attraction initiated by was performed on cDNA samples using the following primers: chemerin for- the Belgian State, Prime Minister’s Office, Science Policy Programming, the ward, 5 -ATGCGACGGCTGCTGATCCCTC-3 , chemerin reverse, 5 - LifeSciHealth (grant no. LSHB-CT-2003-503337) program of the European TTAGCTGCGGGGCAGGGCCTTG-3 ; CXCL10 forward, 5 -GGAAC- Community, the Fonds de la Recherche Scientifique Médicale of Belgium, Télévie, CTCCAGTCTCAGCACC-3 , CXCL10 reverse, 5 -CAGCCTCTGT- and the Fondation Médicale Reine Elisabeth to M. Parmentier. The authors assume GTGGTCCATCC-3 ; CXCL8 forward, 5 -CGATGTCAGTGCATAAA- the scientific responsibility. V. Wittamer was supported by a fellowship of the FIRST- GACA-3 , CXCL8 reverse, 5 -TGAATTCTCAGCCCTCTTCAAAA-3 ; Industrie program of the Walloon Region. D. Communi is Research Associate of the and -actin forward, 5 -GAAGAGCTACGAGCTGCCTGA-3 , -actin Belgian Fonds National de la Recherche Scientifique. reverse, 5 -TGATCTTCATTCTGCTGGGTG-3 . Chemerin PCR condi- The authors have no conflicting financial interests. tions were as follows: 95 C for 3 min, 95 C for 1 min, 60 C for 1 min, 72 C for 2 min (40 cycles), and 72 C for 5 min. CXCL10/IP-10 PCR conditions Submitted: 30 June 2004 were as follows: 95 C for 5 min, 95 C for 30 s, 64 C for 1 min, 72 C for 30 s Accepted: 14 December 2004 (22 cycles), and 72 C for 15 min. 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Role of ChemR23 in directing the migration of myeloid and plasmacytoid dendritic cells to lymphoid organs and inflamed skin

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Copyright © 2005, The Rockefeller University Press
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0022-1007
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10.1084/jem.20041310
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Abstract

BRIEF DEFINITIVE REPORT Role of ChemR23 in directing the migration of myeloid and plasmacytoid dendritic cells to lymphoid organs and inflamed skin 1 2 3 1 William Ver mi, Elena Riboldi, Valer ie Wittamer, Francesca Gentili, 4 1 4 5 Walter Luini, Simona Marrelli, Annunciata Vecchi, Jean-Denis Franssen, 3 2 4 4,6 David Communi, Luisa Massardi, Marina Sironi, Alberto Mantovani, 3 1 2,4 Marc Parmentier, Fabio Facchetti, and Silvano Sozzani 1 2 Department of Pathology and Section of General Pathology and Immunology, University of Brescia, 25123 Brescia, Italy Institut de Recherche Interdisciplinaire en Biologie Humaine et Moléculaire, Campus Erasme, B-1070 Brussels, Belgium Istituto Ricerche Farmacologiche “Mario Negri,” 20157 Milan, Italy Euroscreen S.A., B-6041 Gosselies, Belgium Centro IDET, Institute of General Pathology, University of Milan, 20133 Milan, Italy Chemerin is a chemotactic agent that was recently identified as the ligand of ChemR23, a serpentine receptor expressed by activated macrophages and monocyte-derived dendritic cells (DCs). This paper shows that blood plasmacytoid and myeloid DCs express functional ChemR23. Recombinant chemerin induced the transmigration of plasmacytoid and myeloid DCs across an endothelial cell monolayer. In secondary lymphoid organs (lymph nodes and tonsils), ChemR23 is expressed by CD123 plasmacytoid DCs and by CD1a DC-SIGN DCs in the interfollicular T cell area. ChemR23 DCs were also observed in dermis from normal skin, whereas Langerhans cells were negative. Chemerin expression was selectively detected on the luminal side of high endothelial venules in secondary lymphoid organs and in dermal endothelial vessels of lupus erythematosus skin lesions. Chemerin endothelial cells were surrounded by ChemR23 plasmacytoid DCs. Thus, ChemR23 is expressed and functional in plasmacytoid DCs, a property shared only by CXCR4 among chemotactic receptors. This finding, together with the selective expression of the cognate ligand on the luminal side of high endothelial venules and inflamed endothelium, suggests a key role of the ChemR23/ chemerin axis in directing plasmacytoid DC trafficking. DCs are potent antigen-presenting cells with a subpopulations can be recognized based on their CORRESPONDENCE Silvano Sozzani: unique ability to induce T and B cell responses morphology, location, and phenotypic profile. [email protected] as well as immune tolerance (1, 2). Among M-DC are located in peripheral nonlym- human peripheral blood DCs, at least two dis- phoid tissues at an immature functional state tinct subsets have been defined, based on the where they exert a sentinel function for incom- expression of CD11c, namely CD11c myeloid ing antigens. After antigen uptake, in the con- DCs (M-DC) and CD11c plasmacytoid DCs text of an inflammatory reaction, M-DC leave (P-DC; reference 1). In addition, a minor, still peripheral sites and reach secondary lymphoid poorly characterized population of CD11c organs by lymphatic vessels (5, 6). In contrast, BDCA-3 DCs has been described previously P-DC are typically absent in peripheral tissues (3). M-DC express myeloid markers such as under homeostatic conditions and are believed CD13 and CD33, the stimulatory receptor Ig- to leave the circulation to enter secondary lym- like transcript 1, and can produce high levels of phoid organs through the interaction with high IL-12 (4). Conversely, P-DC have a morphol- endothelial venules (HEVs; references 5, 6). ogy resembling plasma cells; are devoid of However, recently published papers demon- myeloid markers; express high levels of CD4, strated P-DC recruitment also in some inflamed CD62 ligand, and CD123; and can produce skin lesions, such as those associated with sys- high levels of IFN- (5–7). This dichotomy temic lupus erythematosus (LE; references 5, 8), observed in human blood is further extended at in nasal mucosa during allergic reactions, and in the tissue level, where P-DC and many M-DC tumors (for review see reference 5). JEM © The Rockefeller University Press $8.00 509 Vol. 201, No. 4, February 21, 2005 509–515 www.jem.org/cgi/doi/10.1084/jem.20041310 The Journal of Experimental Medicine Differences in distribution and tissue recruitment during inflammatory conditions between M-DC and P-DC may reflect distinct functional properties and be explained by spe- cific migratory mechanisms. Blood DC subsets express a similar pattern of chemotactic receptors, including CCR2, CCR5, CXCR2, CXCR3, CXCR4, and PAFR (9). How- ever, circulating P-DC, in contrast with M-DC, fail to mi- grate in response to inflammatory chemokines, whereas both subsets respond to lymph node–homing chemokines (i.e., CCL19 and CCL21) after maturation (9). Thus, the only chemotactic factor presently known for immature P-DC in vitro is CXCL12, the ligand of CXCR4. However, the mechanisms leading to the in vivo recruitment of P-DC to normal and inflamed tissues are still poorly understood. Chemerin is a novel protein identified as the natural ligand of ChemR23, a previously orphan protein G–coupled receptor expressed by immature DCs and macrophages (10). Chemerin was purified from ovarian cancer ascites and found to correspond to the product of the Tig-2 gene. Chemerin is expressed by many tissues, including spleen and lymph nodes, and is secreted as prochemerin, a poorly active pre- cursor protein. Extracellular proteases convert prochemerin into a full agonist of ChemR23 by proteolytic removal of the last six amino acids (10, 11). Previous work has shown that chemerin induces calcium fluxes, mitogen-activated protein Figure 1. Expression of ChemR23 in leukocytes. PBMCs were isolated kinase activation, and chemotaxis of monocyte-derived im- as described in Materials and methods and stained for ChemR23 expression. mature DCs, whereas LPS- or CD40L-mature DCs do not (a) PBMC subsets were analyzed by FACS analysis. One experiment repre- express functional ChemR23 receptors. The aim of this sentative of four independent experiments is shown. (b) PBMCs were ana- work was to investigate the functional expression of ChemR23 lyzed for the expression of CD14 and CD16 and three populations of mono- in human blood DC subsets. Here, we report that ChemR23 lo cytes were discriminated: CD14 CD16 , CD14 CD16 , and CD14 CD16 . is expressed and functional in M-DC and most importantly ChemR23 expression was investigated in monocyte subsets. The values in P-DC, a property shared only by CXCR4. The ChemR23 represent the mean  SD of positive cells or intensity of membrane stain- ligand, chemerin, is selectively expressed in HEVs and in LE ing (MFI; n  4; *, P  0.05; **, P  0.01 by one-way analysis of variance skin lesions, a prototypic inflammatory condition character- with Tukey post test). The flow cytometry histograms are the result of one experiment representative of four independent experiments. ized by tissue accumulation of P-DC. These data strongly suggest that the ChemR23/chemerin axis may play a key role in regulating the trafficking of P-DC. brane staining (mean fluorescence intensity [MFI]), with the predominant CD14 CD16 population having the lowest RESULTS AND DISCUSSION levels of membrane fluorescence (MFI). Expression and function of ChemR23 in blood The three major blood DC subsets were investigated on MHC class II leukocytes the basis of the expression of specific membrane markers: In a previous work, we reported that chemerin represents a BDCA-1 (M-DC), BDCA-4 (P-DC), and BDCA-3 (3). Fig. new chemotactic factor for monocyte-derived DCs (10). 2 a shows that ChemR23 was expressed by 40% of the cir- Experiments were performed with the aim to characterize culating M-DC population and by virtually all P-DC. DC the expression of ChemR23 in circulating leukocytes. Fig. 1 a maturation (85% CD83 cells), achieved in the presence of shows that ChemR23 expression was mainly present in LPS (M-DC) or influenza virus (P-DC), did not significantly MHC class II cells and in a minor subset of CD3 and affect the percentage of ChemR23 cells, and only induced a CD20 lymphocytes. Within blood mononuclear cells, decrease of the mean channel fluorescence (Fig. 2 a). On the MHC class II cells were the only cells that migrated in re- contrary, ChemR23 was only modestly expressed (30% pos- sponse to chemerin (unpublished data). The expression of itive cells) by BDCA3 DCs, regardless the state of maturation. ChemR23 was further investigated in MHC-II cells. Fig. 1 b Next, we evaluated the ability of chemerin, the ChemR23 shows that ChemR23 was expressed by the three major ligand, to induce DC transmigration across an endothelial cell monocyte subsets, namely CD14 CD16 , CD14 CD16 , monolayer. Chemerin induced a dose-dependent migration lo and CD14 CD16 . However, a statistically significant dif- of immature M-DC and P-DC, with a peak observed at 100 ference among the three populations was found both in pM. Compared with the migration observed in response to an terms of percentage of positive cells and intensity of mem- optimal concentration of CXCL12 (12 nM), chemerin in- 510 RECRUITMENT OF PLASMACYTOID DENDRITIC CELLS BY CHEMERIN | Vermi et al. BRIEF DEFINITIVE REPORT duced the migration of a similar percentage of M-DC, whereas P-DC migration was 50% of that induced by CXCL12. Therefore, chemerin is characterized by potency 100-fold higher (peak chemotactic concentration) and by a similar (M-DC) or slightly reduced (P-DC) efficacy (percent- age of migrated cells) as compared with CXCL12 (Fig. 2 b). Chemotaxis of both M-DC and P-DC to chemerin was com- pletely blocked in the presence of an anti-ChemR23 moAb, proving the involvement of ChemR23 in chemerin-induced DC migration (unpublished data). Of note, chemerin-induced migration of M-DC and P-DC was respectively 1.6- and 3.4- fold more efficient when assessed in transmigration assays than using bare filters. This result suggests that chemerin presenta- tion by endothelial cells is required for optimal interaction with ChemR23 DCs. It was reported previously that CXCR3 ligands increase P-DC migration in response to CXCL12 (12, 13). Therefore, P-DC migration was tested us- ing chemerin in combination with different concentrations of CXCL10 or CXCL12 in the lower wells. P-DC migration to chemerin was never increased by the presence of these two chemokines, indicating that ChemR23 does not cooperate with CXCR3 and CXCR4 for cell migration (unpublished data). Mature M-DC and P-DC did not migrate in response to chemerin (Fig. 2 b), but normally migrated in response to CCL19, one of the CCR7 ligands. It is noteworthy that ma- ture DCs retained high levels of ChemR23 expression, whereas they were unresponsive to the cognate agonist. Thus, the maturation process induces the uncoupling of ChemR23 with no major changes in its membrane expression. All in all, these results show that chemerin represents a new chemotac- tic factor for immature DCs. Most importantly, ChemR23 represents the only chemotactic receptor, in addition to Figure 2. Expression of functional ChemR23 in blood DC subsets. CXCR4, that is active in immature P-DC. Blood DC subsets were isolated as described in Materials and methods and Chemerin belongs to the cathelicidin/cystatin family of stained for ChemR23 expression. Maturation of DCs was achieved by incu- proteins, which includes precursors of bactericidal peptides bation with LPS (BDCA-1 ), influenza virus (BDCA-4 ), or LPS and influ- (cathelicidins), precursors of mediators active on leukocytes enza virus (BDCA-3 ) for 24 h. (a) DC subsets were analyzed by FACS analy- through protein G–coupled receptors (prokininogen, cathe- sis. (b) DC subsets were tested for their ability to migrate across an licidin precursors), as well as cysteine protease inhibitors endothelial cell monolayer in response to chemerin or to an optimal con- (cystatins). Elevated chemerin production was found in centration (12 nM) of CXCL12 (immature DCs) or CCL19 (mature DCs). The ovarian cancer ascites and in the synovial fluids of rheuma- values represent the mean  SD of triplicate data points and are repre- toid arthritis patients (10). Prochemerin transcripts were also sentative of at least five different experiments performed with indepen- dent donors. Note the different scale used in the left versus right panels. found in the skin of psoriatic subjects (14). In the bone stro- (iDC) Immature DCs. (mDC) Mature DCs. *, P  0.05 by paired Student’s t test. mal cell line ST2, the chemerin gene was reported to be in- duced by 1,25-dihydroxy-vitamin D3 (VitD3) and dexa- profiles. For instance, Langerhans cells express CD1a and Lan- methasone (15). Therefore, it is likely that chemerin represents gerin and are exclusively distributed within epithelial surface; an important signal involved in the recruitment of DCs dur- in contrast, the so-called “interstitial DCs,” typically repre- ing inflammation and autoimmune diseases, and at tumor sented by dermal DCs, are mainly CD1a and express DC- sites. The up-regulation of chemerin production by antiin- SIGN and the mannose receptor. In lymphoid organs, DC flammatory signals such as VitD3 and dexamethasone postu- subsets are recognizable in B and T cell compartments and in- lates a role for chemerin in all the conditions characterized clude, respectively, germinal center DCs and Langerhans- by a “type 2/alternative” state of activation, similar to that derived interdigitating DCs in T cell paracortical area (5, 6). present in tumor-associated leukocytes (16). The interfollicular area separates nodular lymphoid compart- ments. The latter is highlighted by the HEVs and populated Expression of ChemR23 by P-DC and M-DC in human tissues by many leukocyte populations, including lymphocytes, macro- At the tissue level, many DC subpopulations can be recog- phages, plasma cells, and at least two distinct DC subpopula- nized based on their morphology, location, and phenotypic tions, namely CD68 DC-SIGN DCs (17) and P-DC (5). JEM VOL. 201, February 21, 2005 511 The expression of ChemR23 was investigated by immu- nohistochemistry in lymph nodes and tonsils. In lymph nodes, numerous ChemR23 cells with a dendritic morphol- ogy were located in the interfollicular compartment (Fig. 3 a). These cells were identified by double immunofluorescence as DC-SIGN (Fig. 3 b) and CD1a cells (Fig. 3 c), two phe- notypic markers that identify distinct nodal myeloid DC pop- ulations (17). On the basis of their phenotype and location, these cells are believed to represent the nodal counterpart of peripheral blood DC-SIGN DCs (18, 19) that are recruited into the lymph nodes from the blood through the HEVs, or a population of dermal DCs migrated to the lymph node. ChemR23 reactivity was also present on CD123 and BDCA-2 P-DC (Fig. 3 d and its inset), either scattered as single cells or in clusters close to HEVs. ChemR23 expres- sion was not restricted to the interfollicular compartment and was also observed in sinus macrophages and in scattered cells within the germinal center (Fig. 3, a and d). This latter popu- lation coexpressed CD68 and represents germinal center macrophages (unpublished data). No reactivity was observed on B and T lymphocytes. These results indicate that ChemR23 expression is retained by P-DC and M-DC after their recruitment to secondary lymphoid tissues. The expression of ChemR23 was also investigated in Figure 3. ChemR23 expression in secondary lymphoid organs and normal skin. A strong reactivity for ChemR23 was identified normal skin. In secondary lymphoid organs, ChemR23 reactivity is ob- in cells located in the upper and intermediate dermis (Fig. 3 e). served in the interfollicular (IF) area and in tingible body macrophages of These ChemR23 cells displayed fusate/dendritic morphol- the germinal center (GC) (a and d). In the interfollicular areas, ChemR23 ogy and coexpressed DC-SIGN (Fig. 3 f) as well as the man- cells present a sparse perivenular distribution (a, black arrowheads), a DC nose receptor (Fig. 3 f, inset), thus corresponding to dermal morphology, and coexpress DC-SIGN (b, white arrowheads) and CD1a DCs (19). On the contrary, ChemR23 expression was never (c, white arrowheads). CD123 ChemR23 plasmacytoid DCs (P-DC; yellow detected in the cutaneous Langerhans cells (Fig. 3, e and f) cells) are also observed in the interfollicular area (IF) in proximity to a CD123 HEV (d), as cluster (d, white asterisk) or as sparse cells coexpressing and in the Langerhans cell–derived paracortical interdigitat- the P-DC–specific marker BDCA-2 (d, inset). In normal skin, ChemR23 ing dendritic cells that accumulate in the lymph node during reactivity is observed in dermal cells with fusate/dendritic morphology (e) dermatopatic lymphadenitis (reference 20 and not depicted). coexpressing DC-SIGN (f, white arrowheads) and mannose receptor (f, inset). These observations clearly indicate that chemerin is unlikely Indirect immunoperoxidase technique was applied (a and e); nuclei were to be a relevant chemotactic factor for the homing of counterstained with hematoxylin. Double immunofluorescence was per- Langerhans cell precursors to the skin, as well as for the mi- formed (b–d and f), using Texas red–conjugated secondary antibodies for gration of Langerhans cells to superficial lymph nodes. anti-ChemR23 (b–d, and e) and anti-BDCA-2 (d, inset) and FITC-conjugated secondary antibodies for anti–DC-SIGN (b and f), anti-CD1a (c), anti-CD123 Expression of chemerin by HEVs in secondary (d), anti-ChemR23 (d, inset), and antimannose receptor (MMR; f, inset). Magnification, 100 (a and d), 200 (e and f), and 400 (b, c, and d and f, insets). lymphoid organs Due to the abundance in ChemR23 cells in secondary lymphoid organs (lymph nodes and tonsils) and normal skin, in close proximity of HEVs that express chemerin suggests the distribution of chemerin was also investigated in these that chemerin represents an alternative/additional signal to tissues. Chemerin reactivity was primarily expressed in the CXCL12 for the recruitment of blood P-DC into lymph interfollicular area of lymph nodes and tonsils (Fig. 4 a), the nodes across HEVs (21). In this respect, it is intriguing that site where the majority of ChemR23 cells also localize. the pattern of chemerin immunoreactivity resembles that The strongest reactivity was observed in endothelial cells observed with an anti-CXCL12 antibody (reference 12 and with cuboidal/cylindrical morphology corresponding to HEVs. unpublished data). In addition to HEVs, chemerin expres- The identity of these chemerin cells was further confirmed sion was also found on sparse spindle cells located in the on the basis of the coexpression of Factor VIII, a specific en- perivenular area (Fig. 4 a). Based on their morphology, this dothelial cell marker (Fig. 4 b, inset), as well as CD123 (Fig. population is likely to represent stromal cells; their precise 4 a, inset) and CLA/Heca452 (Fig. 4 b), two markers also identity is currently under investigation. expressed by tissue P-DC (5). Of note, in HEVs, the highest HEVs were shown to produce CCL21, which accumu- expression of chemerin was observed at the luminal side lates at their luminal side. Other chemokines were detected on (Fig. 4 b). The presence of P-DC positive for ChemR23 the surface of HEVs, although they were produced by non- 512 RECRUITMENT OF PLASMACYTOID DENDRITIC CELLS BY CHEMERIN | Vermi et al. BRIEF DEFINITIVE REPORT HEV cells (e.g., CCL19, CXCL12, and CCL2; references 22, 23). In vitro experiments performed with five different types of endothelial cells of different origin (i.e., cord blood, dermis, iliac artery, and bone marrow) did not show any production of chemerin by endothelial cells both under basal conditions and after cytokine stimulation (TNF, IL-1, LPS, IFN, IFN, and combinations of these agonists; unpublished data). There- fore, it is unknown at the moment whether chemerin expres- sion is a prerogative of a specialized endothelium of an ana- tomical compartment, such as that of HEVs, or if chemerin is produced by stromal cells surrounding HEVs, and subse- quently transported through the fibroblastic reticular cell net- work to HEVs (for review see references 22, 23). Expression of chemerin by dermal endothelial cell of blood vessels in LE Nonresident DCs, including P-DC, accumulate in the skin in many inflammatory conditions (24). Thus, the expression of chemerin was assessed in tissue sections obtained from normal and inflamed skin. Chemerin expression was com- pletely absent in normal skin, including in endothelial cells of the papillary dermis vessels (Fig. 4 c). These data suggest that chemerin is unlikely to be responsible for skin recruit- ment of resident DCs in steady-state conditions. In recent Figure 4. Chemerin expression in secondary lymphoid organs, years, P-DC were directly implicated in the pathogenesis of normal skin, and cutaneous LE lesions. Immunostaining of lymph node LE (5, 8). In LE patients, the number of circulating P-DC is sections shows chemerin reactivity on endothelial cells and sparse periven- reduced, and these cells are present in the skin lesions, sug- ular spindle cells in the interfollicular area (a and b). Chemerin vessels gesting that P-DC are selectively recruited to the skin (8). correspond to HEV labeled by CD123 (a, inset), CLA (b), and Factor VIII (b, inset); Therefore, the expression of chemerin was examined in skin note the preferential expression of chemerin on the luminal side of HEV biopsies from nine cases of LE. Chemerin reactivity was ob- (b, arrowhead). Because CLA and CD123 also stain P-DC, these markers served in six out of the nine cases on the endothelial cells also identify P-DC surrounding HEV (a and b). Chemerin is absent in nor- mal skin (c). Chemerin cells are numerous in the dermis from a LE case (d). lining dermal vessels (Fig. 4, d–f). These endothelial cells did Most of these cells correspond to endothelial cells; in addition, scattered not express podoplanin (Fig. 4 g), a marker of lymphatic en- chemerin cells with fusate morphology are admixed to the lymphoid in- dothelium (25), indicating that they belong to blood vessels. filtrate (d and e). Rare chemerin cells are also noticeable in the epidermis In addition to endothelial cells, rare chemerin cells were (d). Double immunofluorescence demonstrated that, in LE lesions, CD123 also observed in the epidermis and in the dermis. In the epi- P-DC (e, inset, asterisk) are located around chemerin dermal vessels (e, inset; dermis, chemerin reactivity was present in scattered Langer- white arrowheads indicate the luminal side). On serial sections obtained hans cells (Fig. 4 d). In the dermis, sparse chemerin cells from a LE case, chemerin endothelial cells (f) did not coexpress the lym- were admixed to lymphoid infiltrates and displayed a fusate/ phatic endothelial cell marker podoplanin (g). Indirect immunoperoxidase dendritic morphology. These cells were found to corre- technique was applied (a and c–g); nuclei were counterstained with hema- toxylin. Double immunofluorescence was performed (a, inset; b; and e, in- spond to DC-SIGN dendrocytes (reference 26 and unpub- set) using Texas red–conjugated secondary antibodies for antichemerin lished data). Notably, in five out of six positive cases, P-DC (a and b, insets), anti-CLA (b), anti-CD123 (e, inset) and FITC-conjugated were present with variable density within the skin lesions secondary antibodies for anti-CD123 (a, inset), antichemerin (b and e, inset), and double immunofluorescence staining, demonstrating and anti–Factor VIII (b, inset). Magnification, 200 (b–d, f, g, and a and b, that CD123 P-DC are located in close proximity of insets); 400 (a); and 600 (e and its inset). chemerin dermal endothelial cells (Fig. 4 e, inset). This of P-DC across HEVs under steady-state conditions, and in suggests that chemerin local production can direct P-DC to the accumulation of P-DC observed in inflamed tissues. the skin in inflammatory conditions characterized by P-DC accumulation (5). MATERIAL AND METHODS In summary, this report shows that M-DC and P-DC Reagents. Recombinant chemerin was produced and purified as de- scribed previously (10). Human chemokines and cytokines used were: express ChemR23 in vitro and in vivo. Experiments per- CXCL12, CCL19 (PeproTech), IL-1 (Dompè), TNF (BASF Knoll), formed in vitro clearly show that chemerin represents a new IFN (Roussel Uclaf), and IFN (Roferon; Roche). RPMI 1640 was ob- chemotactic factor for blood M-DC and most importantly tained from GIBCO BRL. FCS was obtained from Hyclone. Cytokines for P-DC. The in vivo distribution of chemerin, located at and media were endotoxin free as assessed by the Limulus amoebocyte as- the luminal side of HEVs and inflamed blood dermal vessels, say (BioWhittaker Inc.). All reagents not otherwise specified were obtained strongly suggests that chemerin is involved in the migration from Sigma-Aldrich. JEM VOL. 201, February 21, 2005 513 Peripheral blood DC purification. PBMCs were isolated from buffy as specified before; human iliac artery ECs (27, 28) were grown in E199 with coats by Ficoll gradient (Amersham Biosciences) and peripheral blood 10% FCS, 10% human serum; human dermal microvascular ECs were ob- M-DC and P-DC were magnetically sorted with blood DC Ag (BDCA)-1 tained from PromoCell GmbH and cultured following the manufacturer’s and BDCA-4 cell isolation kits (Miltenyi Biotec), respectively, as described instructions; immortalized human dermal microvascular ECs (29) were previously (9). Blood M-DC and P-DC (10 cells/ml) were cultured in me- grown in E199 with 10% FCS, 10% human serum; human bone marrow mi- dium containing 1,000 U/ml recombinant human GM-CSF (Mielogen; crovascular ECs (30) were grown in E199 as HUVECs. Schering-Plough) and 10 ng/ml IL-13 (a gift from A. Minty, Sanofi Elf Bio Recherches, Labège, France) or 20 ng/ml IL-3 (ProSpec), respectively. Tissues and reagents. Surgical specimens from human tissues including Cells were matured by 24-h incubation with 100 ng/ml LPS (Escherichia coli normal skin (obtained from abdominal plastic surgery), skin biopsies from 055:B5; Sigma-Aldrich), or 20 ng of hemagglutinin/ml inactivated influ- nine cases of LE, reactive tonsils, and lymph nodes (removed for diagnostic enza virus strain A/Moscow/10/99 (a gift from T. De Magistris, Istituto purposes) were analyzed. Each specimen was snap frozen in isopentane pre- Superiore di Sanità, Rome, Italy). BDCA-3 MDC were isolated with cooled to liquid nitrogen temperature and stored at 80 C. Thin cryostat BDCA-3 cell isolation kit (Miltenyi Biotec), cultured in medium contain- sections were air dried overnight at room temperature and fixed in acetone ing 1,000 U/ml rhGM-CSF, 10 ng/ml IL-13, and 20 ng/ml IL-3 and ma- for 10 min before staining. Tissue expression of ChemR23 was evaluated tured by 24-h incubation with 100 ng/ml LPS and 20 ng of hemagglutinin/ by immunohistochemistry using two anti-ChemR23 monoclonal antibod- ml inactivated influenza virus (3). ies, namely 4C7 (IgG2b; dilution 1:15) and 1H2 (IgG2a; 1:15; reference 10). The indirect streptavidin–biotin complex immunoperoxidase tech- Flow cytometry. The expression of ChemR23 was analyzed by FACS us- nique was applied using a biotinylated anti-Ig multilinks secondary antibody ing two monoclonal antibodies (clone 4C7 and 1H2) described previously (1:20 dilution in Tris-HCl buffer, pH 7.4; Biogenex). The 4C7 and 1H2 (10). The expression of ChemR23 on PBMC subsets was analyzed by FACS moAbs properly reacted on frozen sections; no signal was observed in for- using: allophycocyanin-conjugated anti-CD3, PE-conjugated anti-CD20, malin-fixed tissue sections. Tissue expression of chemerin was detected us- and PE-conjugated anti-HLA-DR (Becton Dickinson); and allophycocyanin- ing two mouse monoclonal antibodies (clones 227C and 14G10; both conjugated anti-CD14 and PE-conjugated anti-CD16 (Exalpha). PE-conju- IgG1; dilution 1:30; 2-h incubation), directed against the peptide Cys- gated CD123 (IgG1; clone 9F5) and CD11c (IgG2b; clone S-HCL-3) were QRAGEDPHSFYFPGQFAFS, a common peptide for both active chemerin obtained from Becton Dickinson. Analysis of fluorescence was performed on and prochemerin. a FACStar Plus calibrated with Calibrite beads (Becton Dickinson). Double immunofluorescence stainings were performed using a set of primary antibodies, including CD123 (clone 7G3; mouse IgG2a; dilution Transendothelial migration assay. Transendothelial migration assay was 1:40; BD Biosciences), DC-SIGN (mouse IgG2b; dilution 1:5; BD Bio- evaluated using 24-well Costar Transwell chambers (5 m pore size; Corn- sciences), CLA (clone Heca452; rat IgM; dilution 1:100; BD Biosciences), ing). Human umbilical vein endothelial cells (HUVECs; passage 5) were CD1a (010, IgG1, dilution 1:30; Immunotech), Langerin (provided by subcultured to confluent monolayers on Transwell inserts and precoated with G. Trinchieri, Schering-Plough, Dardilly, France), and mannose receptor gelatin. Monolayers were rinsed with chemotaxis medium (RPMI 1640 con- (clone 3.29B1; mouse IgG1; dilution 1:50). Appropriate Texas red– and taining 10% FCS) before use. 100 l of DC suspension (1–2 10 cells/ml) FITC-conjugated isotype-specific secondary antibodies were used to reveal were seeded in the upper chamber and 600 l of chemoattractant or control the primary antibodies and sections were examined with a fluorescence mi- medium was added to the lower well. The chamber was incubated at 37 C croscope Olympus BX60, equipped with a Nikon digital camera. Anti– in humidified atmosphere in the presence of 5% CO for 4 h. Migrated cells human podoplanin Ab (clone 18H5, dilution 1:30; Abcam) recognizes lym- were recovered from the lower well and counted and the results were ex- phatic endothelium and was tested on frozen samples of breast carcinoma. pressed as the percentage of input cells. In some experiments, DC subsets This work was conducted in accordance with a protocol approved by the were cultured for 24 h in the presence or absence of a maturative stimulus Spedali Civili of Brescia Institutional Ethical Board and informed consent (LPS or influenza virus) and tested for their ability to migrate in response to was obtained from all patients. chemoattractants. PBMCs (10 10 cells/ml; 100 l of cell suspension per We thank M. Cella for the MR moAb. well) migration was tested in the same experimental conditions used for DCs. F. Gentili is supported by “Fondazione Beretta,” and E. Riboldi is supported by Migrated cells were collected and labeled for FACS analysis. “Centro di studio per il trattamento dello scompenso cardiaco.” We also thank the Associazione Italiana per la Ricerca sul Cancro, Ministero dell’Istruzione Università e Chemerin expression in endothelial cells. Chemerin expression was Ricerca, the Association for International Cancer Research (grant no. 04-223), and evaluated by RT-PCR. Total RNA samples (1 g) were reverse transcripted “Fondazione Berlucchi” for financial support to S. Sozzani. This work was also using the SuperScript II Rnase H Reverse Transcriptase (Invitrogen). PCR supported by the Belgian program on Interuniversity Poles of Attraction initiated by was performed on cDNA samples using the following primers: chemerin for- the Belgian State, Prime Minister’s Office, Science Policy Programming, the ward, 5 -ATGCGACGGCTGCTGATCCCTC-3 , chemerin reverse, 5 - LifeSciHealth (grant no. LSHB-CT-2003-503337) program of the European TTAGCTGCGGGGCAGGGCCTTG-3 ; CXCL10 forward, 5 -GGAAC- Community, the Fonds de la Recherche Scientifique Médicale of Belgium, Télévie, CTCCAGTCTCAGCACC-3 , CXCL10 reverse, 5 -CAGCCTCTGT- and the Fondation Médicale Reine Elisabeth to M. Parmentier. The authors assume GTGGTCCATCC-3 ; CXCL8 forward, 5 -CGATGTCAGTGCATAAA- the scientific responsibility. V. Wittamer was supported by a fellowship of the FIRST- GACA-3 , CXCL8 reverse, 5 -TGAATTCTCAGCCCTCTTCAAAA-3 ; Industrie program of the Walloon Region. D. Communi is Research Associate of the and -actin forward, 5 -GAAGAGCTACGAGCTGCCTGA-3 , -actin Belgian Fonds National de la Recherche Scientifique. reverse, 5 -TGATCTTCATTCTGCTGGGTG-3 . Chemerin PCR condi- The authors have no conflicting financial interests. tions were as follows: 95 C for 3 min, 95 C for 1 min, 60 C for 1 min, 72 C for 2 min (40 cycles), and 72 C for 5 min. CXCL10/IP-10 PCR conditions Submitted: 30 June 2004 were as follows: 95 C for 5 min, 95 C for 30 s, 64 C for 1 min, 72 C for 30 s Accepted: 14 December 2004 (22 cycles), and 72 C for 15 min. 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Journal

The Journal of Experimental Medicine – Pubmed Central

Published: Feb 21, 2005

References