CC-Chemokine Ligand 16 Induces a Novel Maturation Program in Human Immature Monocyte-Derived Dendritic Cells This information is current as Paola Cappello, Tiziana Fraone, Laura Barberis, Carlotta of October 1, 2021. Costa, Emilio Hirsch, Angela R. Elia, Cristiana Caorsi, Tiziana Musso, Francesco Novelli and Mirella Giovarelli J Immunol 2006; 177:6143-6151; ; doi: 10.4049/jimmunol.177.9.6143 http://www.jimmunol.org/content/177/9/6143 Downloaded from References This article cites 45 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/177/9/6143.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 1, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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 © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology CC-Chemokine Ligand 16 Induces a Novel Maturation Program in Human Immature Monocyte-Derived Dendritic Cells1 Paola Cappello,*† Tiziana Fraone,* Laura Barberis,‡ Carlotta Costa,‡ Emilio Hirsch,‡ Angela R. Elia,*¶ Cristiana Caorsi,*¶ Tiziana Musso,§ Francesco Novelli,*¶ and Mirella Giovarelli2*¶ Dendritic cells (DCs) are indispensable for initiation of primary T cell responses and a host’s defense against infection. Many proinflammatory stimuli induce DCs to mature (mDCs), but little is known about the ability of chemokines to modulate their maturation. In the present study, we report that CCL16 is a potent maturation factor for monocyte-derived DCs (MoDCs) through differential use of its four receptors and an indirect regulator of Th cell differentiation. MoDCs induced to mature by CCL16 are Downloaded from characterized by increased expression of CD80 and CD86, MHC class II molecules, and ex novo expression of CD83 and CCR7. They produce many chemokines to attract monocytes and T cells and are also strong stimulators in activating allogeneic T cells to skew toward Th1 differentiation. Interestingly, they are still able to take up Ag and express chemokine receptors usually bound by inflammatory ligands and can be induced to migrate to different sites where they capture Ags. Our findings indicate that induction of MoDC maturation is an important property of CCL16 and suggest that chemokines may not only organize the migration of MoDCs, but also directly regulate their ability to prime T cell responses. The Journal of Immunology, 2006, 177: http://www.jimmunol.org/ 6143–6151. endritic cells (DCs)3 are the most important link be- Furthermore, DCs are affected by endogenous mediators released tween the innate and the acquired immune response and by infected, damaged, or inflamed tissues, such as proinflamma- D are involved in the initiation of both types of immunity tory cytokines, prostaglandin, or catabolites from dying cells (5, (1). Immature DC (iDC) precursors exit from the bone marrow, 6). Chemokine receptors have also been shown to be directly in- circulate via the bloodstream to reach their target tissues, and take volved in induction of DC maturation. Specifically, activation up residence at sites of potential pathogen entry in a physiological through CCR5 by Toxoplasma gondii-derived cyclophilin leads to by guest on October 1, 2021 stage that is specialized for Ag capture (1). Tissue injury and in- production of high levels of IL-12 in DCs (7, 8). Little is known, flammation cause dramatic changes throughout the microenviron- on the other hand, about chemokines and DC maturation. We fo- ment, including the release of inflammatory mediators such as cy- cused our attention on CCL16/liver-expressed chemokine’s effects tokines and chemokines that shape protective immune responses because we had demonstrated its potent antitumor activity associ- and culminate in pathogen elimination. Many stimuli are involved ated with recruitment of leukocytes (9). CCL16 is chemotactic for in DC maturation (2). DCs receive information directly from monocytes, lymphocytes, eosinophils, and DCs (for a review, see pathogens via “pattern-recognition receptors” such as the TLRs (3) Zlotnik et al. (10)). It binds CCR1, CCR2, CCR5, and CCR8 (11, and from cytokines released by T cells during their differentiation 12) and has been used to treat established murine tumors. CCL16 toward Th1 or Th2 in peripheral tissues or lymph nodes (LNs) (4). expressed by adenoviruses (13, 14) or as recombinant fusion pro- tein (15) induces accumulation of T cells and DCs at the tumor site *Center for Experimental Research and Medical Studies, San Giovanni Battista Hos- pital, Turin, Italy; †Department of Clinical and Biological Sciences, University of and in draining LNs. Turin, Orbassano, Italy; ‡Department of Genetics, Biology, and Biochemistry, §De- Our previous data have shown that CCL16 costimulates mac- ¶ partment of Public Health and Microbiology, and Department of Medicine and Ex- rophage cytotoxic and Ag-presenting functions in the mouse (16). perimental Oncology, University of Turin, Turin, Italy CCL16 released by lamina propria-infiltrating macrophages elicits Received for publication November 22, 2005. Accepted for publication August 14, 2006. massive recruitment of immune reactive cells in ulcerative colitis The costs of publication of this article were defrayed in part by the payment of page (17). These results led us to study the effects of CCL16 on human charges. This article must therefore be hereby marked advertisement in accordance DCs derived from monocytes (MoDCs) in the presence of GM- with 18 U.S.C. Section 1734 solely to indicate this fact. CSF and IL-4. We report here that human CCL16 is a potent mat- 1 This work was supported by the Italian Association for Cancer Research, the Italian Ministry for Education, the Universities and Research, Fondi incentivazione della uration factor for MoDCs through differential use of its four re- Ricerca di Base and Progetti di Ricerca di Interesse Nazionale, and Compagnia San ceptors and an indirect regulator of Th cell differentiation. We Paolo, Special Project Oncology. analyzed the expression of costimulatory molecules and chemo- 2 Address correspondence and reprint requests to Professor Mirella Giovarelli, De- kine receptors and confirmed their functional efficiency. We also partment of Experimental Medicine and Oncology, Corso Raffaello 30, 10125 Torino, Italy. E-mail address: [email protected] evaluated the ability of MoDCs induced to mature by CCL16 3 Abbreviations used in this paper: DC, dendritic cell; CCL16/mDC, MoDC induced (CCL16/mDCs) to induce alloactivation of T cells and take up Ag. to mature in the presence of CCL16; iDC, immature DC; LN, lymph node; mDC, DC Our findings suggest that chemokines may not only organize the induced to mature in the presence of TNF-␣ plus IL-1␤; MFI, mean fluorescence intensity; MoDC, monocyte-derived DC; PLC, phospholipase C; PTX, pertussis migration of DCs but also directly regulate their ability to prime T toxin. cell responses. Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 6144 MoDC MATURED BY CCL16 CAN STILL TAKE UP Ag Materials and Methods Mixed lymphocyte reaction CCL16 source T cells were purified on a nylon wool column (Robbins Scientific) and ϫ 5 The human recombinant CCL16 used in these experiments (PeproTech) is placed in 96-well plates at 1 10 cells/well with allogeneic iDCs and differentially mature DCs, after 24 h of stimulation with CCL16 or TNF-␣ a 11.2-kDa protein of 97-aa residues produced in Escherichia coli from a and IL-1␤, in increasing concentrations (150–10, 000). After 4 days, 1 ␮Ci DNA sequence encoding the mature human CCL16 protein sequence 3 (Q26-Q120). Its endotoxin level is Ͻ0.1 ng/␮g(1EU/␮g) as determined of tritiated [ H]TdR (Amersham Biosciences) was added to each well and incubation was prolonged for an additional 18 h. Cells were directly col- by the Limulus Amebocyte Lysate assay. lected with a CellHarvester (Packard Instrument) in UNIfilter plates (Pack- ard Instrument) and [3H]TdR uptake was quantitated (TopCount micro- Generation and maturation of MoDCs plates scintillation counter; Packard Instrument). All tests were performed Human PBMCs were isolated from venous blood of voluntary healthy in triplicate. donors by Ficoll-Paque density gradient centrifugation (Pharmacia Bio- Supernatants from T cells cultured with iDCs, CCL16/mDCs, or mDCs at ratio 20:1 T:DC were harvested after 48 h and analyzed by ELISA for tech). Monocytes were enriched with an isolation kit (Miltenyi Biotec). ␥ The resulting preparations were consistently Ͼ90% CD14ϩ as determined IFN- and IL-4 production (both R&D Systems) in accordance with the by FACSCalibur (BD Biosciences). To prepare iDCs, the enriched mono- manufacturer’s protocol. ϫ 6 cytes were incubated in 6-well culture plates (5 10 cells/3 ml/well) in Cytokine detection AIM V medium (Invitrogen Life Technologies) supplemented with 100 ng/ml GM-CSF and 50 ng/ml IL-4 (both PeproTech) for 6 days. On days Production of cytokines and chemokines in the supernatants was evaluated 2 and 4, two-thirds of the culture medium were replaced by fresh medium with a commercial RayBio Human Cytokine Array V (Tebu-bio) in accor- containing GM-CSF and IL-4. iDCs were resuspended as 1 ϫ 106 cells/ml dance with the manufacturer’s protocol. The membranes were exposed to in AIM V supplemented or not with CCL3, CCL4, CCL14, and CCL16 (all x-ray films and signals were detected with a film developer (Kodak Hy- from PeproTech) at 1000 and 100 ng/ml, as indicated, or human TNF-␣ (50 perfilm; Amersham Biosciences).