Unique Regulation of CCL18 Production by Maturing Dendritic Cells Marisa Vulcano, Sofie Struyf, Patrizia Scapini, Marco Cassatella, Sergio Bernasconi, Raffaella Bonecchi, Angelica This information is current as Calleri, Giuseppe Penna, Luciano Adorini, Walter Luini, of October 4, 2021. , Jo Van Damme and Silvano Sozzani J Immunol 2003; 170:3843-3849; ; doi: 10.4049/jimmunol.170.7.3843 http://www.jimmunol.org/content/170/7/3843 Downloaded from

References This article cites 68 articles, 27 of which you can access for free at: http://www.jimmunol.org/content/170/7/3843.full#ref-list-1 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Unique Regulation of CCL18 Production by Maturing Dendritic Cells1

Marisa Vulcano,* Sofie Struyf,† Patrizia Scapini,‡ Marco Cassatella,‡ Sergio Bernasconi,* Raffaella Bonecchi,*¤ Angelica Calleri,* Giuseppe Penna,ሻ Luciano Adorini,ሻ Walter Luini,* Alberto Mantovani,*¶ Jo Van Damme,† and Silvano Sozzani2*¤

Dendritic cells (DC) orchestrate the trafficking of lymphocytes by secreting with different specificity and function. Chemokines are produced at higher levels by mature DC. This study shows that CCL18 is one of the most abundant chemokines produced by immature DC. In contrast to all other chemokines investigated to date, CCL18 was selectively down-regulated during the maturation process induced by LPS, TNF, CD40 ligand, Staphylococcus aureus Cowan I, Candida albicans, and influenza virus.

IL-10 and vitamin D3, two known inhibitors of DC differentiation and function, strongly promoted CCL18 secretion, whereas ␥ IFN- , a costimulator of DC function, inhibited its production. IL-10 also induced CCL18 secretion in blood myeloid DC. No Downloaded from CCL18 secretion was observed in blood plasmacytoid DC. The opposite pattern of regulation was observed for CCL20, a pro- totypic inflammatory . CCL18 was found to be a chemotactic factor for immature DC. Therefore, CCL18 may act as a chemotactic signal that promotes the colocalization of immature DC with naive T lymphocytes in an IL-10-dominated envi- ronment with the consequent generation of T regulatory cells. These characteristics suggest that CCL18 may be part of an inhibitory pathway devoted to limiting the generation of specific immune responses at peripheral sites. The Journal of Immu-

nology, 2003, 170: 3843Ð3849. http://www.jimmunol.org/

endritic cells (DC)3 are potent APC with a unique ability and ) present at the site of DC activation will determine to induce T and responses (1–3). DC reside in an the quality of the response generated (17–20). D immature state in peripheral tissues where they exert a Chemokines are a large family of chemotactic proteins that play sentinel function for incoming Ags (2, 4). Following encounter a crucial role in regulating leukocyte composition in inflamed tis- with Ags, in the context of an inflammatory situation, or upon sues (6, 8, 21–23). DC secrete high levels of several chemokines direct stimulation by specific pathogens, DC undergo a process of (24, 25). DC-derived chemokines are believed to contribute to the maturation that enhances their APC functions and promotes their recruitment of precursor cells and immature DC at the peripheral by guest on October 4, 2021 migration to the draining lymph nodes (5, 6). In the secondary sites of inflammation (6, 8, 22) and within the lymph nodes, where lymphoid organs, mature DC prime naive T cells (7–9). The proper they play a role in T and B cell localization and the DC-T cell localization of DC in secondary lymphoid organs and their recruit- interaction (2, 8). Chemokine production is usually associated with ment at sites of inflammation in response to chemotactic stimuli DC maturation both in vitro and in vivo (24–26). However, a are critical for an optimal immune response (10, 11). limited number of chemokines, such as -derived che- DC are not only potent initiators of immune responses, they also mokine/CCL22, TARC/CCL17, and PARC/DC-CK1/macrophage play an important regulatory role. DC can induce either a Th1 inflammatory protein-4/CCL18, are secreted in a constitutive man- cytotoxic response or favor a Th2 humoral-polarized response (12–14). Furthermore, DC can function as tolerogenic cells in re- ner by both immature -derived DC and blood myeloid sponse to self or environmental Ags (5, 15, 16). Recent evidence DC (24, 25, 27–29) (M. Vulcano, unpublished observations). indicates that the microenvironment (e.g., type of Ag, pathogen, Whereas several studies have investigated the up-regulation of CCL22 and CCL17 in maturing DC, the regulation and the role of CCL18 in DC biology are poorly understood. CCL18 is a chemo- ‡ *Istituto di Ricerche Farmacologiche Mario Negri, Milan, Italy; Department of Pa- kine active on naive T cells and B lymphocytes with no known thology, Section of General Pathology, University of Verona, Verona, Italy; †Rega Institute, University of Leuven, Leuven, Belgium; §Section of General Pathology and receptor identified to date and no rodent homologues (28, 30–33). Immunology, University of Brescia, Brescia, Italy; ¶Section of General Pathology, CCL18 mRNA expression was reported in , DC, normal University of Milan, Milan, Italy; and ʈBioXell, Milan, Italy lung, pneumonitis-affected lungs, germinal centers of regional Received for publication August 29, 2002. Accepted for publication February 3, 2003. lymph nodes and tonsils, atherosclerotic plaques, inflamed liver, The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance septic rheumatoid , and dermis of contact with 18 U.S.C. Section 1734 solely to indicate this fact. patients (28, 30, 31, 33–38). Recent work has identified CCL18 as 1 This work was supported in part by Istituto Superiore di Sanita`, Special Projects on the major chemokine produced by tumor-associated AIDS, Ministero Istruzione Università e Ricerca (Cofin 2001), Consiglio Nazionale in ovarian carcinoma (39). The aim of this study was to investigate Delle Ricerche Target Project Biotechnology, the Ministero della Sanita`, the Centro di Eccellenza IDET, and the European Commission. the regulation of CCL18 production at both the mRNA and protein 2 Address correspondence and reprint requests to Dr. Silvano Sozzani, Istituto di levels in human DC at different stages of maturation. In addition, Ricerche Farmacologiche Mario Negri, via Eritrea 62, 20157 Milan, Italy. E-mail CCL18 chemotactic activity on DC migration was investigated. address: [email protected] The results reported here outline a unique pattern of regulation for 3 Abbreviations used in this paper; DC, ; CD40L, CD40 ligand; Dex, dexamethasone; M-DC, myeloid DC; P-DC, plasmacytoid DC; SAC, Staphylococcus CCL18 in maturing DC, and we propose that this chemokine is an aureus Cowan I; VitD3, vitamin D3. inhibitory signal in the control of the immune response.

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 3844 CCL18 AND DC

Materials and Methods ELISA Cell culture media and reagents An in-house sandwich ELISA for CCL18 was developed by coating plates with polyclonal anti-human CCL18 Ab (R&D Systems, Abingdon, U.K.). The following reagents were used for tissue culture: pyrogen-free saline A second polyclonal anti-human CCL18 Ab (PeproTech, Rocky Hill, NJ) (S.A.L.F., Bergamo, Italy), RPMI 1640 (Biochrom, Berlin, Germany), and was used to detect bound immunoreactivity (39). Similar results were ob- aseptically collected FCS (HyClone Laboratories, Logan, UT). All reagents tained using CCL18/PARC Duoset kit (R&D Systems). CCL20 and IL- contained Ͻ0.125 endotoxin units/ml, as checked by the Limulus amebo- 12(p75) were measured in the cell-free supernatants by ELISA, using spe- cyte lysate assay (Microbiological Associates, Walkersville, MD). LPS cific Abs as described previously (24, 44). from Escherichia coli strain 055:B5 (LPS) was obtained from Difco (De- troit, MI), and Staphylococcus aureus Cowan I (SAC) was purchased from RT-PCR Calbiochem (San Diego, CA). Inactivated influenza virus strain A/Mos- cow/10/99 was a gift from Dr. T. De Magistris (Istituto Superiore di Sanita`, Total RNA was reverse transcribed to cDNA. Subsequently, the cDNAs Rome, Italy). Candida albicans was obtained as previously described (40). obtained were PCR amplified using specific primer pairs for VitD3 receptor Ј Ј Ј Dexamethasone (Dex), PGE2, and 1,25-dihydroxyvitamin D3 (VitD3) were (sense, 5 -CATCGGCATGATGAAGGAGTT-3 ; antisense, 5 -CATCA obtained from Sigma-Aldrich (St. Louis, MO). Human rGM-CSF was a gift TGTCTGAAGAGGTGATACAG-3Ј). The oligonucleotide primers used from Novartis (Milan, Italy). Human IL-13 was a gift from Dr. A. Minty in RT-PCR experiments were designed on distinct to exclude an (Sanofi Elf Bio Recherches, Labe`ge, France). Human TNF-␣ was obtained amplification of possible contaminating genomic DNA in the RNA sam- from BASF/Knoll (Ludwighafen, Germany). Human rIL-10 was purchased ples. Amplification of ␤-actin cDNA was also conducted as an internal from Schering-Plough (Milan, Italy), and human rIFN-␥ was obtained from control for the efficiency of RNA extraction and RT. PCR products were Roussel-UCLAF (Paris, France). Neutralizing mAb against TNF-␣ (B.154.2) separated and visualized on an ethidium bromide-stained agarose gel. and an isotype-matched control were provided by Dr. G. Trinchieri (Schering- Plough, Dardilly, France). Anti-IL-10 mAb (23738.11) was obtained from Migration assay R&D Systems (Minneapolis, MN) and was used as previously described (41).

DC migration was evaluated using a 48-well microchemotaxis chamber Downloaded from ␣ Anti-TNF- and anti-IL-10 mAbs had undetectable endotoxin levels by the technique. Briefly, 27 ␮l of chemokine or control medium (RPMI 1640 Limulus amebocyte lysate assay. with 1% FCS) was added to the lower wells of the chemotaxis chamber Monocyte-derived DC preparation (Neuroprobe, Pleasanton, CA) (43). Fifty microliters of cell suspension (1.5 ϫ 106/ml) were seeded in the upper chamber. The two compartments DC were generated as previously described (24). Briefly, highly enriched were separated by a 5-␮m pore size polyvinylpyrrolidone polycarbonate blood monocytes (Ͼ95% CD14ϩ) were obtained from buffy coats (through filter (Neuroprobe). The chamber was incubated for 90 min at 37°Cina

the courtesy of the Centro Trasfusionale, Ospedale Civile Formalori, Ma- humidified atmosphere in the presence of 5% CO2. At the end of the in- genta, Italy) by Ficoll (Biochrom) and Percoll gradients (Pharmacia Fine cubation, filters were removed and stained, and five high power oil im- http://www.jimmunol.org/ Chemicals, Uppsala, Sweden). Monocytes were cultured for 6 days at 1 ϫ mersion fields (ϫ1000) were counted per well. Results are expressed as the 106/ml in six-well tissue culture plates (Falcon; BD Biosciences, Franklin mean of three replicates Ϯ SD of a single experiment representative with Park, NJ) in RPMI 1640 supplemented with 10% FCS, 50 ng/ml GM-CSF, at least three independent donors. and 20 ng/ml IL-13. Where indicated, DC were further cultured in the presence of 100 ng/ml LPS and 20 ng/ml TNF for 48 h or as otherwise Statistical analysis specified. CD40 ligand (CD40L)-transfected J558L cells or mock-trans- Statistical significance between the experimental groups was determined fected control cells were cultured with DC at a 1:4 ratio. Incubation of DC using paired Student’s t test. with the J558L mock-transfected cells did not induce cell maturation or chemokine production (data not shown). Results Peripheral blood DC purification and culture Inhibition of CCL18 secretion by maturing DC by guest on October 4, 2021 PBMC were isolated from buffy coats by Ficoll gradient (Pharmacia Bio- In the first set of experiments the kinetics of CCL18 production tec, Uppsala, Sweden), and peripheral blood myeloid (M-DC) and plas- during DC maturation were investigated. Immature DC were macytoid (P-DC) DC were magnetically sorted with BDCA-1 and washed, and maturation was induced using an optimal concentra- BDCA-4 cell isolation kits (Miltenyi Biotec, Bergisch Gladblach, Ger- many), respectively, as previously described (42) to a purity of 95–98%. tion (100 ng/ml) of LPS. As expected on the basis of previous Blood M-DC and P-DC (2 ϫ 104 cells/well) were cultured in 96-well plates work, immature DC release CCL18 in a constitutive manner (28, (Costar, Cambridge, MA) in RPMI 1640 culture medium supplemented 31). Fig. 1 shows that CCL18 is released at high levels (387 Ϯ 66 ␮ with 5% FCS, 2 mM L-glutamine, 50 g/ml gentamicin, 1 mM sodium ng/106 cell, after 48-h incubation; n ϭ 15), making this protein one pyruvate, and 1% nonessential amino acids plus 1000 U/ml GM-CSF and 10 ng/ml IL-4 (BD PharMingen, San Diego, CA) or 20 ng/ml IL-3 (BD of the most highly expressed chemokines by this cell type (24). PharMingen), respectively. Where indicated, cells were stimulated with 10 LPS stimulation did not alter the levels of CCL18 secretion up to ng/ml IL-10 or 1 ␮g/ml LPS, SAC (1/5000), CD40L-transfected J558L 8 h. Thereafter, LPS strongly suppressed CCL18 production cells at a ratio of 4:1, 6 ␮g/ml CpG oligonucleotides 2006 (MGW Biotech, (109 Ϯ 21 ng/106 cells at 48 h; n ϭ 15). The kinetics of chemokine Ebersberg, Germany), and 20 ng/ml hemagglutinin-inactivated influenza secretion under both basal and stimulated conditions were paral- virus strain A/Moscow/10/99. After 24 h of culture, supernatants were collected, and chemokine concentrations were measured by ELISA. leled by specific mRNA expression as detected by Northern blot analysis (data not shown and see below). Fig. 1 shows that CCL20, Northern blot analysis a proinflammatory chemokine active on memory T cells and im- DC total RNA was extracted by the guanidinium thiocyanate or TRIzol mature DC (45–49) that was investigated in parallel experiments, method, blotted, and hybridized as previously described (43). Probes were was produced in a detectable manner only by LPS-stimulated DC. labeled using the Megaprime DNA labeling system (Amersham Interna- 32 CCL20 became detectable after 8-h incubation, and its production tional, Little Chalfont, U.K.) with [ P]dCTP (3000 Ci/mmol; Amersham 6 International). Membranes were prehybridized at 42°C in Hybrisol (Oncor, increased linearly up to 48 h, reaching 1.7 Ϯ 0.1 ng/10 DC (n ϭ Gaithersburg, MD) and hybridized overnight with 1 ϫ 106 cpm/ml 32P- 4). In the same experimental conditions other inflammatory che- labeled probe. Membranes were then washed three times with 2ϫ SSC (1ϫ mokines (CXCL8, CCL2, and CCL3) were also found to be up- ϭ SSC 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) at room tem- regulated (data not shown). perature for 10 min, twice with 2ϫ SSC/1% SDS at 60°C for 20 min, and then with 0.1ϫ SSC for 5 min before being autoradiographed using Kodak Different protocols for inducing DC maturation in vitro were XAR-5 films (Eastman Kodak, Rochester, NY) and intensifier screen at employed. DC were stimulated in the presence of 100 ng/ml LPS Ϫ80°C. The CCL20-specific probe was obtained as previously described (pathogen-derived agonist), 20 ng/ml TNF-␣ (a proinflammatory (44). The CCL18-specific probe was obtained by RT-PCR amplifying the ), or CD40L-transfected cells (a T cell-derived signal). full-length cDNA reported sequence (GenBank accession no. 4506830) Ј Ј After 48 h of culture, DC maturation was assessed in all cultures with specific primers (sense, 5 -ATCATGAAGGGCCTTGCAGCTGC-3 ; ϩ ϩ antisense, 5Ј-TCAGGCATTCAGCTTCAGGTCGC-3Ј) and confirmed by as the percentage of CD83 and DC-LAMP cells (always Ͼ85% sequencing. and Ͼ80%, respectively; data not shown). Fig. 2 shows that both The Journal of Immunology 3845 Downloaded from

FIGURE 1. Opposite kinetics of CCL18 and CCL20 production in ma- turing DC. DC (106/ml) were cultured in the absence (control) or the pres- ence of LPS (100 ng/ml). Supernatants were collected at different time http://www.jimmunol.org/ points and were tested for chemokine production by specific ELISA. Re- sults are expressed as the mean (ϮSD) of 4–15 independent experiments. .p Ͻ 0.05 vs control ,ء

CD40L and LPS stimulated CCL20 expression, with TNF-␣ being FIGURE 2. Different regulation of CCL18 and CCL20 production by maturing DC. DC (106/ml) were incubated in the absence (Ϫ) or the pres- a marginal, if any, agonist for this chemokine. In contrast, all three ence of 100 ng/ml LPS, 20 ng/ml TNF, or CD40L (4:1 ratio of DC to maturation agents inhibited CCL18 production after 48 h of stim- CD40L transfectants) for 48 h. Supernatants were evaluated for the pres- by guest on October 4, 2021 Ͼ Ͼ ulation, although with different potencies: CD40L LPS TNF ence of chemokines by ELISA. The top panels show chemokine levels. (71, 65, and 43% inhibition, respectively). mRNA levels, as eval- Results are the average determinations (ϮSD) of eight independent exper- uated by Northern blot analysis using specific probes, paralleled iments. The lower panels report Northern blot analysis of total RNA (10 p Ͻ ,ء .the chemokine protein expression (Fig. 2, top and bottom, lower ␮g/lane) purified from DC in one experiment representative of four .((p Ͻ 0.01 (vs control (Ϫ ,ءء ;panels). Down-regulation of CCL18 production during DC matu- 0.05 ration was also observed when other pathogen-derived maturing agents (SAC, C. albicans, and influenza virus) were used (Table I). was increased further after 48 h (Fig. 3B). Despite the fact that Maturation of DC is associated with TNF secretion (50). To assess IL-10 is known to inhibit LPS function in phagocytic cells, includ- the contribution of this potential autocrine loop in LPS- and ing DC, IL-10 could not reverse LPS-induced inhibition of CCL18 CD40L-induced CCL18 inhibition, experiments were performed production (Fig. 3B). In the same experimental conditions CCL20 in the presence of a TNF-␣-blocking mAb (51). Results obtained with three different donors showed no effect of the anti-TNF Ab on CCL18 production by both immature and mature DC (data not Table I. Down-regulation of CCL18 production by pathogen-derived shown). maturing stimulia

Divergent regulation of CCL18 production by IFN-␥ and IL-10 CCL18 (% inhibition) The maturation and function of DC are controlled by pro- and DC Treatment 24 h 48 h anti-inflammatory cytokines. For instance, IFN-␥ is known to pro- mote IL-12 production by mature DC, whereas IL-10 inhibits DC LPS 46.2 Ϯ 9.6b 76.7 Ϯ 5.2b maturation and IL-12 production (4, 12, 52). IL-12 production by SAC 56.5 Ϯ 9.5b 69.9 Ϯ 2.5b Ϯ b Ϯ b DC is pivotal to orient T cells to a Th1-polarized phenotype, C. albicans 47.2 2.5 71.5 3.5 Influenza virus 19.6 Ϯ 16 51.5 Ϯ 10.5b whereas in the presence of IL-10, T cells with Th2/T regulatory functions are generated (20). Fig. 3A shows that reduced levels of a DC were treated with LPS (100 ng/ml), SAC (1/5000), C. albicans (100 ␮g/ml), ␥ and 20 ng/ml hemagglutinin-inactivated influenza virus strain A/Moscow/10/99. Cul- CCL18 are secreted by immature DC in the presence of IFN- (46 ture supernatants were collected after 24 and 48 h of stimulation, and CCL18 levels and 56% inhibition ( p Ͻ 0.01) at 24 and 48 h, respectively). The were detected by ELISA. The data shown represent the percent inhibition of CCL18 Ϯ degree of the inhibition was almost comparable to that observed production with respect to immature DC (0% inhibition corresponds to 170 26 and 312 Ϯ 47 ng/106 DC CCL18 at 24 and 48 h, respectively). Results are expressed as following LPS-induced maturation. No additive effect was ob- the mean (Ϯ SD) of three independent experiments. The ability of these stimuli to served when LPS and IFN-␥ were used together. In contrast, IL-10 promote DC maturation was assessed in parallel as the expression of CD83 at 24 h of culture (% CD83ϩDC, 89 Ϯ 5, 78 Ϯ 3, 82 Ϯ 6, and 29 Ϯ 8 for LPS, SAC, C. strongly increased CCL18 secretion. The effect was already ap- albicans, and virus, respectively). parent after 24 h of stimulation (60% increase over control) and b p Ͻ 0.05 vs iDC. 3846 CCL18 AND DC Downloaded from

6 FIGURE 4. Induction of CCL18 in immature DC by VitD3. A,DC(10 / ml) were incubated in medium alone (control) or in the presence of VitD3 (10Ϫ7 M) for 4 h and then stimulated with LPS (100 ng/ml) for 48 h. Supernatants were tested by ELISA. Results are the average determinations FIGURE 3. Regulation of CCL18 production by IFN-␥ and IL-10. DC 6 (ϮSD) of three to seven independent experiments. The average (100%) (10 /ml) were cultured with LPS (100 ng/ml), IFN-␥ (500 U/ml), or both http://www.jimmunol.org/ CCL18 (by immature DC) and CCL20 (by mature DC) productions were LPS and IFN-␥ (A) and with IL-10 (20 ng/ml) or IL-10 and LPS (B). 366 Ϯ 55 and 1.8 Ϯ 0.6 ng/106 DC, respectively. B, Total RNA was Supernatants were collected at different time points and were tested for isolated from immature DC and mature DC that had been stimulated with CCL18 production by ELISA. Protein levels are expressed as the mean LPS or CD40L for 48 h. The expression of VitD receptor (VD3R) mRNA p Ͻ 0.01; #, p Ͻ 0.05 (vs 3 ,ء .ϮSD) of three independent experiments) p Ͻ ,ء .was analyzed by RT-PCR as described in Materials and Methods control (Ϫ)). 0.01 vs control (Ϫ); #, p Ͻ 0.05 vs LPS.

(40%) inhibited CCL20 release by LPS-stimulated DC (Fig. 4A)as expression was induced by IFN-␥ and inhibited by IL-10 (data not well as the release of CCL22 (24) (data not shown). by guest on October 4, 2021 shown) as previously reported (48). In contrast, both Dex and PGE2 decreased CCL18 release by Recent evidence indicates that endogenous IL-10 production immature DC of ϳ55% (n ϭ 4; Fig. 5A). A similar degree of might regulate DC maturation and functions (41) To rule out a inhibition was observed when DC were cultured with either LPS or possible role of endogenous IL-10 in the constitutive CCL18 re- CD40L regardless of the presence of the two inhibitors (Fig. 5A). lease by immature DC, experiments were performed in the pres- Although both Dex and PGE2 had a negative effect on DC matu- ence of a blocking IL-10 Ab. Results obtained with three different ration and IL-12 production (Fig. 5B), these treatments did not DC cultures excluded a role of endogenous IL-10 in CCL18 pro- reverse maturation-induced inhibition of CCL18 release. Parallel duction (data not shown). experiments showed that CCL20 production in LPS-stimulated DC

was inhibited in the presence of PGE2, whereas it was resistant to Regulation of CCL18 secretion by inhibitors of DC maturation the effect of Dex (data not shown) as previously reported (55). As

The results obtained with IL-10 were extended to other drugs expected, Dex, PGE2, and VitD3 under the experimental condi- known to affect different aspects of DC maturation and function, tions used blocked in a similar manner (Ͼ80%) IL-12 production by DC cultured with LPS or CD40L (Fig. 5B and data not shown). such as VitD3, corticosteroids, and PGE2. VitD3 and Dex block DC maturation (e.g., CD83 expression and IL-12 production) and Production of CCL18 by blood M-DC and P-DC inhibit chemokine production (e.g., CCL22, CXCL8, and CCL2), (24, 53) (M. Vulcano, unpublished observations). On the other The studies on CCL18 production were extended to circulating DC hand, PGE2 does not affect the acquisition of a mature DC phe- subsets. Neither M-DC nor P-DC produced CCL18 in a constitu- notype, but does inhibit IL-12 production (54). tive manner; following in vitro culture with a number of maturing

Similar to the observations with IL-10, VitD3 did not modify agonists, including LPS, SAC, influenza virus, CpG, and CD40L; LPS-induced inhibition of CCL18 production. However, in the ab- or in the presence of VitD3 (data not shown). However, in agree- sence of a maturation signal, VitD3 induced CCL18 production in ment with the results obtained with monocyte-derived DC, IL-10 a dose-dependent manner (Fig. 4A). The effect was already evident was able to selectively up-regulate CCL18 production in M-DC, Ϫ9 Ͻ Ϫ7 Ͻ Ϯ Ϯ 6 at 10 M(p 0.05) and peaked at 10 M(p 0.01) VitD3 with 19 4 and 103 17 ng/10 M-DC after 24- and 48-h cul- ϭ (data not shown). The expression of VitD3 receptors was equally ture, respectively (n 3). On the contrary, no CCL18 production well observed in immature and mature DC by PCR analysis (Fig. was observed by IL-10-stimulated P-DC (data not shown). 4B). It must be noted that a certain degree of donor-to-donor vari- ability was observed in these experiments; some donors (three of Migration of immature DC to CCL18 seven) were only weakly responsive to the effect of the drug (data Finally, the ability of CCL18 to induce chemotaxis of DC was not shown). The reason for this variability is currently under in- investigated. Fig. 6 shows that CCL18 was able to induce chemo- vestigation. Under the same culture conditions VitD3 partially taxis of immature DC with a bell-shaped dose-response curve. At The Journal of Immunology 3847

was induced by any of the three CCL18 preparations, indicating that the still unknown CCL18 receptor must be inactivated/down- regulated during the DC maturation process (data not shown).

Discussion DC are located at the interface of innate and specific immunity. At their immature stage DC reside in nonlymphoid organs, and after the capture of an Ag within an inflammatory context, DC undergo maturation and migrate to secondary lymphoid organs (2–6, 8). The DC contribution to the onset and evolution of the immune response is represented by their ability to release large amounts of chemokines, some of them with a predominant proinflammatory function (e.g., CCL2 and CXCL8) (21, 56), and others more in- volved in leukocyte positioning and cell-cell interaction (e.g., CCL19 and CCL22) (10, 24, 57). In this study we investigated the regulation of CCL18 (a chemotactic factor for naive T lympho- cytes and CD38Ϫ B cells (28, 58)) in both immature and mature DC. The secretion of CCL20, an inflammatory chemokine active on memory T cells and B lymphocytes (47, 49), was examined in parallel studies for comparison. This study reports two main find- Downloaded from ings. First, CCL18 and CCL20 undergo opposite regulation during DC maturation. CCL18 is down-regulated during DC maturation, FIGURE 5. CCL18 and CCL 20 production is differentially regulated whereas the maturation process induces CCL20 as well as all other 6 during alternative pathways of DC maturation. DC (10 /ml) were incubated chemokines investigated to date (59). Second, CCL18 is a chemo- in the presence of Dex (10Ϫ5 M) or PGE (10Ϫ5M) for 4 h and then 2 tactic agonist for immature DC.

stimulated with LPS (100 ng/ml), CD40L, or medium alone (control) for http://www.jimmunol.org/ CCL18 is released at high levels by immature DC (387 Ϯ 66 48 h. Supernatants were tested by ELISA. Results are the average deter- 6 minations (ϮSD) of eight independent experiments. Protein production in ng/10 DC in 48 h). Therefore, CCL18, together with CCL22, CCL17, and CXCL8, represents one of the most abundantly se- the absence of Dex or PGE2 was considered as 100% of chemokine pro- duction and corresponded to 380 Ϯ 6.4 ng/ml CCL18 in DC control (A), creted chemokines by DC (24, 25). CCL18 inhibition was detect- and 0.25 Ϯ 0.02 and 0.68 Ϯ 0.02 ng/ml IL-12 in LPS-stimulated DC and able after 8 h and was observed with all maturation agonists used (p Ͻ 0.05 vs respective no ad- (i.e., LPS, TNF, CD40L, SAC, C. albicans, and influenza virus ,ء .(CD40L-stimulated DC, respectively (B dition (Ϫ). No production of IL-12 was observed in DC control superna- and in the presence of IFN-␥, a costimulator of DC functions (12). tants (not shown). Previous studies performed by RT-PCR reported a limited induc- tion of CCL18 mRNA in 24-h LPS-stimulated DC (25). However, by guest on October 4, 2021 the peak of chemotactic response the number of migrated DC was in the present study the decrease in CCL18 protein secretion was similar to that observed in the presence of an optimal concentration paralleled by the inhibition of CCL18 mRNA levels, as evaluated of CCL3 (43). Because the optimal CCL18 concentration slightly by Northern blot, a more quantitative analysis than RT-PCR. The varied (between 100 and 300 ng/ml) using two different commer- reason for this discrepancy is at present uncertain. To our knowl- cially available CCL18 preparations, results were confirmed using edge, this is a unique feature, since only one other chemokine, natural purified CCL18 (39). Fig. 6 shows that natural CCL18 was CXCL13, was shown to be selectively inhibited at the mRNA level also active as a chemotactic agonist for immature DC, with an by CD40L, but not by LPS or TNF stimulation (60). Incubation of optimal concentration of 100 ng/ml. No migration of mature DC immature DC with Dex and PGE2 also resulted in a partial de- crease in CCL18 secretion. The blocked acquisition of the DC mature phenotype by these two agents did not reverse CCL18 in- hibition. Conversely, up-regulation of CCL18 secretion by imma-

ture DC was observed in the presence of VitD3 and IL-10, two known suppressors of DC maturation and function (52, 53). VitD3 is an immunosuppressive agent active in autoimmune diseases and graft rejection models by inhibiting Ag-induced T cell prolifera- tion, cytokine production, and Th1 development (61, 62). IL-10 is an anti-inflammatory cytokine produced in a constitutive way at the mucosal sites and involved in the inhibition of inflammatory and immune reactions in many pathological conditions, including tumors (63). It is interesting to note that Dex was reported to en- hance IL-4-induced expression of CCL18 in human monocytes (31). In the same experimental system CCL18 was induced by FIGURE 6. Migration of immature DC in response to CCL18. Imma- IL-10. CCL18 was recently identified as the most abundant che- ture DC (iDC) were assayed for their migratory capacity toward recombi- mokine in human ovarian ascites. CCL18 was produced by tumor- nant (R&D Systems or PeproTech) and purified natural CCL18 (39). The associated macrophages, very likely in response to tumor- and migration assay was performed as indicated in Materials and Methods. Results are expressed as the mean (ϮSD) of three replicates of a single macrophage-derived IL-10 (39). The regulation of CCL18 produc- experiment and are representative of at least three independent donors. tion in DC reported in this study strengthens the concept that Basal migration, against medium (12 Ϯ 2 cells), was subtracted from the CCL18 is a mediator of the alternative activated (or type II) APC data. The dashed line indicates the number of migrated cells in response to response (64). Cytokines and drugs associated with polarized type Ͻ ءء Ͻ ء 100 ng/ml CCL3. , p 0.05; , p 0.01 vs medium (no chemokine). II responses (e.g., IL-4, IL-10, corticosteroids, and VitD3) induce 3848 CCL18 AND DC an alternative activation program with distinct functional proper- mokine have defects in lymphocyte homing and dendritic cell localization. ties. These functionally polarized cells play a key role in the sub- J. Exp. Med. 189:451. 12. Moser, M., and K. M. Murphy. 2000. Dendritic cell regulation of TH1-TH2 version of adaptive immunity and in inflammatory circuits that development. Nat. Immunol. 1:199. promote tolerance and tumor progression (65). 13. Langenkamp, A., M. Messi, A. Lanzavecchia, and F. Sallusto. 2000. Kinetics of There is evidence that stimulation of naive T lymphocytes with dendritic cell activation: impact on priming of TH1, TH2 and nonpolarized T cells. Nat. Immunol. 1:311. immature DC induces the generation of IL-10-producing T regu- 14. Kalinski, P., C. M. Hilkens, E. A. Wierenga, and M. L. Kapsenberg. 1999. T-cell latory cells (20). Since CCL18 is chemotactic for both immature priming by type-1 and type-2 polarized dendritic cells: the concept of a third DC and naive T cells, CCL18 production by immature DC could signal. Immunol. Today 20:561. 15. Garza, K. M., S. M. Chan, R. Suri, L. T. Nguyen, B. Odermatt, S. P. Schoenberger, promote the interaction of these two cell types. Finally, a recent and P. S. Ohashi. 2000. Role of -presenting cells in mediating tolerance and study by Nibbs et al. (66) showed that CCL18, in addition to being . J. Exp. Med. 191:2021. a T cell chemoattractant, exhibits antagonist activity for CCR3, a 16. Steinman, R. M., and M. C. Nussenzweig. 2002. Avoiding horror autotoxicus: the importance of dendritic cells in peripheral T cell tolerance. Proc. Natl. Acad. Sci. chemotactic receptor for eosinophils and basophils. Taken to- USA 99:351. gether, these findings strongly suggest that at nonlymphoid sites 17. Pulendran, B., J. Banchereau, E. Maraskovsky, and C. Maliszewski. 2001. Mod- CCL18 may not act as a proinflammatory chemokine, but, rather, ulating the immune response with dendritic cells and their growth factors. Trends Immunol. 22:41. it may represent a negative mechanism to limit the onset of im- 18. Granucci, F., C. Vizzardelli, E. Virzi, M. Rescigno, and P. Ricciardi-Castagnoli. mune reactions. CCL18 antagonistic activity for CCR3 may serve 2001. Transcriptional reprogramming of dendritic cells by differentiation stimuli. to antagonize local production of proallergic chemokines. Further- Eur. J. Immunol. 31:2539. 19. Huang, Q., N. Liu do, P. Majewski, A. C. Schulte le, J. M. Korn, R. A. Young, more, the recruitment of naive T cells by immature DC may favor E. S. Lander, and N. Hacohen. 2001. The plasticity of dendritic cell responses to a tolerogenic condition. However, in the presence of a severe in- pathogens and their components. Science 294:870. flammatory situation, DC will be induced to mature with the con- 20. Jonuleit, H., E. Schmitt, G. Schuler, J. Knop, and A. H. Enk. 2000. Induction of Downloaded from 10-producing, nonproliferating CD4ϩ T cells with regulatory prop- sequent down-regulation of CCL18 production. This CCL18 ho- erties by repetitive stimulation with allogeneic immature human dendritic cells. meostatic function might be increased in the presence of J. Exp. Med. 192:1213. immunosuppressive, anti-inflammatory cytokines, such as IL-10, 21. Baggiolini, M. 1998. Chemokines and leukocyte traffic. Nature 392:565. 22. Sallusto, F., C. R. Mackay, and A. Lanzavecchia. 2000. The role of chemokine or during immunosuppressive pharmacological treatments, as in receptors in primary, effector, and memory immune responses. Annu. Rev. Im- the case of VitD3 or corticosteroid administration. Therefore, munol. 18:593. CCL18 production by immature DC could be part of a default 23. Horuk, R. 2001. Chemokine receptors. Cytokine Rev. 12:313. http://www.jimmunol.org/ 24. Vulcano, M., C. Albanesi, A. Stoppacciaro, R. Bagnati, G. 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