Junction Proteins Population Expressing Langerin and Tight
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The Journal of Immunology ␣  A Major Lung CD103 ( E)- 7 Integrin-Positive Epithelial Dendritic Cell Population Expressing Langerin and Tight Junction Proteins1 Sun-Sang J. Sung,2*‡ Shu Man Fu,*‡ C. Edward Rose, Jr.,* Felicia Gaskin,‡† Shyr-Te Ju,*‡ and Steven R. Beaty*‡ Dendritic cells (DC) mediate airway Ag presentation and play key roles in asthma and infections. Although DC subsets are known to perform different functions, their occurrence in mouse lungs has not been clearly defined. In this study, three major lung DC populations have been found. Two of them are the myeloid and plasmacytoid DC (PDC) well-characterized in other lymphoid ␣  high high organs. The third and largest DC population is the integrin E (CD103) 7-positive and I-A CD11c -DC population. This population was found to reside in the lung mucosa and the vascular wall, express a wide variety of adhesion and costimulation ␣ ؉  ␣ molecules, endocytose avidly, present Ag efficiently, and produce IL-12. Integrin E 7 DC ( E-DC) were distinct from intra- epithelial lymphocytes and distinguishable from CD11bhigh myeloid and mPDCA-1؉B220؉Gr-1؉ PDC populations in surface marker phenotype, cellular functions, and tissue localization. Importantly, this epithelial DC population expressed high levels of the Langerhans cell marker Langerin and the tight junction proteins Claudin-1, Claudin-7, and ZO-2. In mice with induced airway hyperresponsiveness and eosinophilia, ␣E-DC numbers were increased in lungs, and their costimulation and adhesion molecules were up-regulated. These studies show that ␣E-DC is a major and distinct lung DC population and a prime candidate APC with the requisite surface proteins for migrating across the airway epithelia for Ag and pathogen capture, transport, and presentation. They exhibit an activated phenotype in allergen-induced lung inflammation and may play significant roles in asthma pathogenesis. The Journal of Immunology, 2006, 176: 2161–2172. endritic cells (DC)3 are the predominant APC type (1, 2) node, plus two additional lymph node populations that are either and play critical roles in airway antigenic and patho- CD8ϪDEC-205high or CD8lowDEC-205low. The DEC- genic responses (3–6). DC processes extend into the ep- 205highCD8low population expresses Langerin, and is postulated to D ϩ ithelia to form an I-A reticular structure for Ag capture (7–9). In represent the matured form of Langerhans cells that has migrated the homeostatic state, DC turnover occurs rapidly in the airway to the lymph node. In that regard, a similar subset of epithelium with a 2-day half-life (10). Enhanced lung DC migra- CD11chighCD40highCD8␣int DC population that also expresses tion to the draining lymph nodes is initiated by TLR ligands or ␣  ␣  1 1 and E 7 has been found in the skin-draining lymph node Ag-specific T cells (11–15) with the appearance of Ag-loaded DC (17). Besides these conventional DC subsets with varying lineage in the thoracic lymph node within 6 h and peaking between 2 and or tissue origin, an IFN-␣-producing PDC has been described in 3 days (11–13). mice (18, 19). These cells are CD11cϩI-AϩB220ϩGr-1ϩ. A new The major DC hallmark is their potent Ag presentation capabil- marker described for PDC (20, 21) will facilitate the isolation and ity. DC have been classified according to their surface marker phe- further characterization of PDC in lungs. Functionally, lung PDC notype and functions into myeloid, lymphoid, and plasmacytoid has been shown to be important in suppressing antigenic responses DC (PDC) (1). In mice, five populations of lymph node DC have in lungs (22). Lung DC isolated in mouse, rat, and human (23–26) been described (16). Among them are the double-negative ϩ Ϫ Ϫ ϩ ϩ ϩ are MHC class II but mostly exhibit an immature phenotype. CD4 CD8 CD11b myeloid DC, the CD4 CD11b myeloid high ϩ Mouse lung DC characterized thus far belong to the CD11b DC, and the CD8 lymphoid DC present in both spleen and lymph ϩ ϩ myeloid population (27–29). No CD8␣ lymphoid DC or CD4 myeloid DC characterized in spleen and lymph node (16, 30) has been reported in lungs. However, very low numbers of PDC with *Department of Internal Medicine and †Department of Psychiatric Medicine, and ‡University of Virginia Specialized Center of Research in Systemic Lupus Erythem- GR-1 and B220 expression have been detected in the lung alveolar atosus, University of Virginia School of Medicine, Charlottesville, VA 22908 septa by immunohistochemistry (28). Recent studies of lung cells Received for publication September 21, 2005. Accepted for publication December in excised lungs and respiratory tracts also showed that PDC is 2, 2005. present in low numbers in both CD11cϩ and CD11cϪ populations The costs of publication of this article were defrayed in part by the payment of page identified by a PDC-specific mAb, although these populations have charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. not been examined in detail (29). In addition to the myeloid and high low Ϫ 1 This work was supported in part by National Institutes of Health Grants HL070065, PDC subset, a rapidly migrating CD11b CD11c I-A popu- HL65344, AR45222, and AI36938. lation has been found in the respiratory tract. 2 Address correspondence and reprint requests to Dr. Sun-Sang J. Sung, Division of DC subsets have been shown to produce distinct cytokines, me- Rheumatology and Immunology, Department of Internal Medicine, Box 800412, Uni- diate different Th subset responses, present autoantigens through versity of Virginia Health Sciences Center, Charlottesville, VA 22908. E-mail: [email protected] their ability to internalize apoptotic cells, regulate antigenic re- 3 ␣ ␣  ϩ sponses, and migrate differently in response to chemokines (1, 22, Abbreviations used in this paper: DC, dendritic cell; E-DC, integrin E 7 DC; PDC, plasmacytoid DC. 31, 32). To understand the regulation of immune responses in the Copyright © 2006 by The American Association of Immunologists, Inc. 0022-1767/06/$02.00 ␣  ϩ 2162 INTEGRIN E 7 LUNG MUCOSAL DC normal and diseased states in lungs, it is critical to characterize cluded from analysis by 7-aminoactinomycin D staining. Flow cytometry subset DC and study their functions separately. In this report, lung results were analyzed by the program FlowJo (Tree Star). CD11cϩI-Ahigh-DC populations enriched by anti-CD11c-magnetic Immunofluorescence and confocal microscopy microbeads have been resolved into two populations, an integrin ␣  ϩ ␣ high E 7 DC population ( E-DC) and a CD11b population Lung tissues were fixed-inflated in 0.7% paraformaldehyde as described (CD11bhigh-DC), based on their integrin ␣  , CD11b, I-A, and (33), equilibrated in 30% sucrose, and embedded in OCT. Sections (5 m) E 7 ␥ ␥ CD11c cell surface expression. Immunofluorescence microscopy were extracted with 0.3% Triton X-100, blocked with anti-Fc RII/Fc RIII ␣ mAb 2.4G2 and serum, and stained with primary and secondary Ab. Con- showed that the E-DC were mainly localized in the lung epithelia focal microscopy was performed on a Zeiss LSM510 assembly with 488, and in the arteriolar wall of naive and immunized mice. Flow 546, and 633 excitation lines. Data were compiled using the software pro- cytometry analyses further showed that these DC constitute a ma- vided by the manufacturer. jor population of the I-AhighCD11chigh-DC in lungs. ␣E-DC are distinct from the CD11bhigh lung DC in surface phenotype, func- DC pinocytosis of FITC-dextran tional characteristics, and lung localization site, and they are Lung CD11cϩ DC were suspended at 1 ϫ 106 cells/ml, preincubated at clearly different from intraepithelial lymphocytes and PDC iso- 37°C for 15 min with and without 5 mg/ml mannan, and allowed to pino- lated by anti-mPDCA-1 magnetic microbeads. Functionally, cytose FITC-dextran (0.5 mg/ml) for the indicated lengths of time. Cold PBS was used to stop the uptake and in cell washing. After mAb staining, ␣ ϩ E-DC internalize FITC-dextran avidly, stimulate anti-CD3 and FITC-dextran uptake was measured by flow cytometry for I-AhighCD103 , Ag-dependent T cell proliferation efficiently, and produce IL-12 I-AhighCD11bhigh, and I-AϩSiglac-Fϩ cells representing ␣E-DC, upon stimulation by TLR ligands. In mice with induced asthma, CD11bhigh-DC, and macrophages, respectively. lung ␣E-DC numbers and costimulation and adhesion molecule surface expression increased. Importantly, these DC express tight DC stimulation of T cell proliferation junction proteins Claudin-1, Claudin-7, and ZO-2 that will allow Spleen CD4ϩ T cells from DO11.10 transgenic mice were purified to 98% them to traverse the lung epithelia readily. Furthermore, ␣E-DC purity by depletion with magnetic microbeads conjugated with anti-CD19, express Langerin, which suggest that they are similar to CD8ϩ CD11c, CD8, and DX5 mAb. Proliferation was performed as described (35). T cells were stimulated with sorted ␣E-DC or CD11bhigh-DC and lymphoid-derived DC and Langerhans cells. The results show for either 2 g/ml anti-CD3 mAb or 5 M OVA323–339 peptide. Total spleno- the first time that ␣E-DC constitute a major DC population resid- cytes irradiated for 25 Gy were used as control APC. ing in the lung mucosa, are competent in key DC functions, reside at specific mucosal locations, and exhibit an activated phenotype in Microarray analysis of ␣E-DC and CD11bhigh-DC mRNA asthma-induced mice. They may play key roles in airway antigenic Magnetic bead-purified CD11cϩ cells from lung digests were stained responses and asthma. with anti-IA-FITC, anti-CD103-PE, anti-CD11c-allophycocyanin, anti-CD11b-Cy7-allophycocyanin, and 7-aminoactinomycin D and ϩ ϩ Materials and Methods sorted for the I-AhighCD103 CD11c CD11blow (␣E-DC) and the I-AhighCD103ϪCD11cϩCD11bhigh (CD11bhigh-DC) populations in the Materials live cell gate in a two-way sort.