CCL9 Is Secreted by the Follicle-Associated Epithelium and Recruits Dome Region Peyer's Patch CD11b+ Dendritic Cells This information is current as Xinyan Zhao, Ayuko Sato, Charles S. Dela Cruz, Melissa of October 1, 2021. Linehan, Andreas Luegering, Torsten Kucharzik, Aiko-Konno Shirakawa, Gabriel Marquez, Joshua M. Farber, Ifor Williams and Akiko Iwasaki J Immunol 2003; 171:2797-2803; ; doi: 10.4049/jimmunol.171.6.2797 http://www.jimmunol.org/content/171/6/2797 Downloaded from References This article cites 32 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/171/6/2797.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 CCL9 Is Secreted by the Follicle-Associated Epithelium and Recruits Dome Region Peyer’s Patch CD11b؉ Dendritic Cells1 Xinyan Zhao,2* Ayuko Sato,* Charles S. Dela Cruz,† Melissa Linehan,* Andreas Luegering,3‡ Torsten Kucharzik,3‡ Aiko-Konno Shirakawa,§ Gabriel Marquez,¶ Joshua M. Farber,§ Ifor Williams,‡ and Akiko Iwasaki4* The follicle-associated epithelium (FAE) secretes chemokines important in the recruitment of various cell types including CCL20 MIP-3␣). CCL20 is chemotactic to the CD11b؉ dendritic cells (DCs) distributed in the subepithelial dome regions of the Peyer’s) patches, and mice deficient in the receptor for CCL20, CCR6, have been reported to be devoid of the CD11b؉ DCs in the dome regions. Here, we describe another chemokine specifically secreted from the FAE of mouse Peyer’s patches, CCL9 (MIP-1␥, CCF18, MRP-2). By in situ hybridization, we demonstrated that CCL9 mRNA was expressed by the FAE but not by the villus epithelium. At the protein level, CCL9 was detected on the FAE and on extracellular matrix structures within the dome regions of the Peyer’s patches. By RT-PCR, we demonstrated that one of the putative receptors for CCL9, CCR1, was expressed by the Downloaded from Peyer’s patch CD11b؉ DCs and in a chemotaxis assay, CD11b؉ DCs migrated toward CCL9. To compare the abilities of the chemokines CCL20 and CCL9 to recruit CD11b؉ DCs to the dome regions, we examined the in vivo distribution of these cells in CCR6-deficient, CCL9-blocked wild type, or CCL9-blocked CCR6-deficient mice. To our surprise, using a sensitive immunoflu- orescence analysis, we observed that CD11b؉ DCs were present in the dome regions of the CCR6-deficient mice. In contrast, Ab neutralization of CCL9 in vivo resulted in significant reduction of the CD11b؉ DC number in the subepithelial dome regions of /Peyer’s patches of both wild type and CCR6 ؊/؊ mice. Taken together, these results demonstrate an important role of CCL9 in http://www.jimmunol.org .CD11b؉ DC recruitment to the dome regions of mouse Peyer’s patches. The Journal of Immunology, 2003, 171: 2797–2803 he Peyer’s patches are lymphoid organs located in the in the interfollicular regions. The latter two populations share sim- small intestine, which serve as the major sites for gener- ilar functional characteristics, namely, that they both secrete IL-12 ation of immunity to intestinal pathogens. In previous p70 upon bacterial stimulation and induce predominantly Th1 re- T 5 ϩ studies, three separate populations of dendritic cells (DCs) were sponses in naive TCR transgenic CD4 T cells in vitro (2). identified with distinct anatomical distribution pattern and diverse The chemokines that coordinate the localization of these DC sub- functions (1, 2). The DCs that specifically localize in the subepi- sets have been proposed to include CCL20 and CCL21/CCL19. The by guest on October 1, 2021 thelial dome regions of the Peyer’s patches express the CD11b follicle-associated epithelium (FAE) overlying the dome regions of molecule and secrete mainly IL-10 upon in vitro stimulation with the Peyer’s patches was found to express high levels of CCL20 (1, 3). CD40L or with killed Staphylococcus aureus. The second DC sub- Further, the dome region CD11bϩ DCs have a unique capacity to ␣␣ ϩ set expresses the CD8 molecule and is localized in the T cell- migrate toward CCL20 (1). In contrast, CD8␣ DCs and the DN DCs rich interfollicular regions of the Peyer’s patches. The third DC both express CCR7 mRNA and migrate toward CCL21 expressed in ␣ subset lacks the expression of CD11b and CD8 , thus named dou- the T cell regions of the Peyer’s patches. The importance of CCL20 ble negative (DN) DCs, and is found in both the dome region and in CD11bϩ DC recruitment to the dome regions of the Peyer’s patches was later reported in CCR6-deficient mice (3, 4). Departments of *Epidemiology and Public Health and †Internal Medicine, Yale Uni- In the process of analyzing other chemokines that are specifi- ‡ versity School of Medicine, New Haven, CT 06520; Department of Pathology and cally secreted by the FAE, we have identified the chemokine Laboratory Medicine, Emory University School of Medicine, Atlanta, GA 30322; §Inflammation Biology Section, Laboratory of Clinical Investigation, National Institutes CCL9. CCL9, also known as macrophage inflammatory protein of Allergy and Infectious Diseases, Bethesda, MD 20892; and ¶Departamento de Immu- (MIP)-1␥, MIP-related protein (MRP)-2 and CCF18, is a mouse nologı´a y Oncologı´a, Centro Nacional de Biotecnologı´a, Consejo Superior de Investiga- ciones Cientı´ficas, Universidad Auto´noma de Madrid, Cantoblanco, Madrid, Spain CC chemokine independently cloned by three different groups (5– Received for publication March 21, 2003. Accepted for publication July 8, 2003. 7). Among CC chemokine family members, the mouse CCL9 The costs of publication of this article were defrayed in part by the payment of page shares 45, 24, and 20% aa sequence identity with mouse CCL6 charges. This article must therefore be hereby marked advertisement in accordance (C10), CCL3 (MIP-1␣), and CCL4 (MIP-1␤), respectively (7). with 18 U.S.C. Section 1734 solely to indicate this fact. Unlike chemokines such as CCL3 and CCL4, whose expression is 1 This work was supported by a Burroughs Wellcome Fund Career Award in Bio- induced by inflammatory stimuli, CCL9 was shown to be consti- medical Sciences. tutively expressed by a wide variety of tissues, and relatively high 2 Current address: Department of Microbiology, 203 C Johnson Pavilion, University ␮ of Pennsylvania Medical Center, Philadelphia, PA. concentrations (1 g/ml) of CCL9 has been detected in the circu- 3 Current address: Department of Medicine, University of Muenster, Albert- lation of normal mice (7). The CCL9 protein can be secreted from Schweitzer-Str. 33, D-48129 Muenster, Germany. Langerhans’ cells, DCs (8) and macrophage cell lines (6), and has 2ϩ ϩ 4 Address correspondence and reprint requests to Dr. Akiko Iwasaki, Department of been shown to induce chemotaxis and Ca flux in CD4 T cell Epidemiology and Public Health, 60 College Street, LEPH 716, New Haven, CT clones (5) and in splenic CD4ϩ T cells (8). However, the precise 06520. E-mail: [email protected]. expression of CCL9 in vivo has not been examined thus far. 5 Abbreviations used in this paper: DC, dendritic cell; DN, double negative; FAE, follicle-associated epithelium; WT, wild type; MIP, macrophage inflammatory pro- In this study, we demonstrated the expression of CCL9 on the tein; DIG, digoxygenin. FAE, but minimally on villus epithelium, at the mRNA and at the Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 2798 CCL9 SECRETION BY PEYER’S PATCH ASSOCIATED EPITHELIUM protein levels. A putative receptor for CCL9, CCR1 was found to purified and cloned into pGEM-T-EASY vector (Promega, Madison, WI), be highly expressed by the CD11bϩ DCs. The CD11bϩ DCs iso- and sequenced to confirm 100% match to the previously published se- lated from the Peyer’s patches exhibited migratory activity to quence (GenBank accession number NM_011338). The restriction en- zymes, NdeI and XbaI, were used to linearize the plasmid for the genera- CCL9. Moreover, a careful examination of the CCR6-deficient tion of anti-sense and sense probes, respectively. The transcript yield and mice, generated from four separate laboratories, using an amplified integrity of the probes were determined by spectrophotometric analysis and immunofluorescence technique revealed the presence of CD11bϩ electrophoresis, respectively. Mouse tissues were first fixedfor3hin4% DCs in the dome regions of their Peyer’s patches. These results paraformaldehyde in 0.14 M Sorenson’s phosphate buffer and then for overnight in 4% paraformaldehyde/30% sucrose in 0.14 M Sorenson’s suggested that a CCL20/CCR6-independent mechanism exists for ϩ phosphate buffer. Fixed tissues were snap-frozen in OCT compound and the recruitment of CD11b DCs to the dome regions. Finally, Ab- stored at Ϫ80°C. Sections of 7 ␮m thickness were cut and placed onto blocking of CCL9 in either the CCR6-deficient mice or wild type poly-L-lysine (Sigma-Aldrich, St. Louis, MO)-coated glass slides and in (WT) mice resulted in a significant reduction in the number of the situ hybridization was performed as previously described (11).