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. Alberto Mantovani, 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/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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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 chemokines 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 chemokine. 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: 3843–3849. http://www.jimmunol.org/ endritic cells (DC)3 are potent APC with a unique ability and cytokines) present at the site of DC activation will determine to induce T and B cell responses (1–3). DC reside in an the quality of the T cell 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 macrophage-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 monocyte-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 monocytes, 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 arthritis, and dermis of contact hypersensitivity 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 macrophages 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, dendritic cell; 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 exons 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.