Junction Proteins Population Expressing Langerin and Tight

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 intraepithelial 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. Total RNA was extracted immediately TLR ligands were purchased from InvivoGen and have been tested to be with an RNeasy kit (Qiagen), and good quality RNA samples were LPS-free by the manufacturer. All mAb with and without fluorophore con- obtained based on the profiles of 28S and 18S RNA fractionated on an jugation were purchased from eBioscience except the following: anti-CD4, Agilent Bioanalyzer and the 5Ј to 3Ј ratios of housekeeping genes in ␤ Affymetrix gene chip analysis. Gene expression levels in the two DC integrin 7, SiglacF, CD54, CD49e, and TCR mAb were obtained from BD Pharmingen; anti-F4/80 mAb was from Caltag Laboratories; anti-CD205 populations were probed with the Affymetrix Gene-Chip mouse genome mAb was from Serotec; anti-mPDCA-1 mAb was from Miltenyi Biotec; 430 2.0 array by the University of Virginia Biomolecular Facility. high anti-Claudin-1, Caludin-7, and ZO-2 rabbit polyclonal Ab were from Three independent pairs of ␣E-DC and CD11b -DC from different Zymed Laboratories; and rabbit anti-Langerin Ab was from Imgenex. isolations were analyzed by the same chip batch. The cell intensity files Alexa dye-conjugated secondary Ab were from Molecular Probes. ELISA provided by the Affymetrix MAS program were further normalized and kits for IFN-␥, IL-12 p70, and IFN-␣ determination were from Endogen/ background was subtracted to provide gene expression levels using the Pierce, R&D Systems, and PBL Biomedical Labs, respectively. FITC-dex- program dChip (36). tran and Saccharomyces cerevisiae mannan were purchased from Sigma-Aldrich. Real-time PCR analysis of DC mRNA DC purification Aliquots of total RNA from FACS-sorted DC subsets were reverse transcribed by the Advantage RT-for-PCR kit (BD Clontech). Real-time PCR Lung single-cell suspensions were prepared essentially as described (33). was performed in a Bio-Rad iCycler Thermal Cycler using Sybr green Total lung DC preparations were obtained by CD11c-magnetic microbead fluorescence as the readout, and data were analyzed by the iCycler program selection according to the manufacturer’s protocol (Miltenyi Biotec). PDC provided (Bio-Rad). PCR conditions were as follows: 94°C for 22 s, 62°C were isolated from lung digests by magnetic cell sorting with anti- for 30 s, and 72°C for 30 s for 39 cycles; 94°C for 22 s, 62°C for 30 s, and mPDCA-1 magnetic microbeads. Macrophages were isolated by anti-F4/ 72°C for 5 min for 1 cycle. Melt curves were obtained by increasing the 80-magnetic microbeads. Pure ␣E-DC and CD11bhigh-DC populations temperature from 65°C to 95°C in 0.5°C increments for 10 s. The primer ϩ were isolated from CD11c cells by sorting on a FACSVantage SE flow sequences were generated by the program Primer 1, and the primers were cytometer. synthesized by IDT. The cDNA amplified, 5Ј-primer sequence, 3Ј-primer sequence, and product size, respectively, are as follows: Claudin-1, Mice and immunization 5Ј-CCCAGTGGAAGATTTACTCCTAGT-3Ј,5Ј-TGCAAAGTACTGT Ј Ј The immunization of BALB/cByJ mice for asthma induction was per- TCAGATTCAGC-3 , 151 bp; Claudin-7, 5 -GCTTCTTAGCCATGTTT Ј Ј Ј formed as described (33) with two notable exceptions: 1) animals were GTCG-3 ,5-CAAACTCGTACTTAACGTTCATGG-3 , 213 bp; ZO-2, ϩ Ј Ј Ј adoptively transferred i.v. with 2.5 ϫ 106 splenic CD4 DO11.10 trans- 5 -CTGCTCAATTACACTCAGTGTTCT-3 ,5-GGCTGTAAAAAGAT Ј Ј genic T cells two days before DC sensitization; and 2) mice were sensitized GAGAACAGGT-3 , 171 bp; Langerin, 5 -ACAAGGAGCAAAGTAGGA Ј Ј Ј intratracheally with bone marrow-derived DC (34) instead of splenic DC. GGTTCT-3 ,5-CAGATCTGTCATTCAGTTGTTTGG-3 , 178 bp; and ␤ Ј Ј Ј The protocols in this study have been approved by the University of Vir- -actin, 5 -CTCTTTTCCAGCCTTCCTTCTTGG-3 ,5-CTCCTTCTG Ј ginia Institutional Use and Care of Animals Committee. CATCCTGTCAGCAAT-3 , 181 bp. Flow cytometry analysis Statistics Lung and lymph node cells were blocked with anti-Fc␥RII/Fc␥RIII mAb The mean and SD of multiple trials were calculated by Excel (Microsoft). 2.4G2, stained with fluorophore-conjugated mAb, and analyzed by flow Statistical significance using Student’s t test was determined by the pro- cytometry using FACSCalibur (BD Biosciences). Dead cells were ex- gram SlideWrite plus (Advanced Graphics Software). The Journal of Immunology 2163

Results contains an extensive epithelial surface, it is likely that the ␣E-DC ␣E-DC and CD11bhigh-DC are the two major populations of found in other epithelial tissue such as rat intestinal mucosa (37, I-Ahigh lung DC 38) would constitute a significant lung DC population or perhaps population two of the I-Ahigh DC (Fig. 1Ab). This hypothesis was Previous studies have identified the CD11bhigh-DC as the only found to be indeed the case. The lung I-Ahigh population with major DC populations in lungs (27–29). However, when anti- slightly higher CD11c but lower I-A expression (population 2 in CD11c-magnetic bead-enriched DC from lung digests were ana- ϩ Fig. 1Ab) was found to be integrin ␣E (␣E population in Fig. 1B, lyzed by flow cytometry, two different I-Ahigh DC populations b and c) but did not express lymphocyte Peyer’s patch high endo- based on I-A and CD11c expression levels were found (Fig. 1Ab, thelial venule adhesion molecule 1 (␣ ␤ ; data not shown). This populations 2 and 3). The mean fluorescence intensities for pop- 4 7 population has a low to intermediate surface CD11b expression ulations 2 and 3 were: I-A, 217 and 388, and CD11c, 153 and 22, (Fig. 1Bb). However, a small population of these ␣E-DC (Ϸ5% of respectively. A third major population comprising ϳ50% of the ϩ ␣E cells) was CD11bhigh representing perhaps activated ␣E-DC. CD11cϩ cells and with high autofluorescence and CD11c staining Large numbers of the two I-Ahigh DC types could be isolated by (Fig. 1, Ab, population 1, and B, b–d, population M) are pulmo- magnetic bead sorting. ␣E-DC and CD11bhigh-DC each comprised nary macrophages, the further characterization of which will be ϳ1% of the total lung cell digests (0.7–1.6% and 1.1–1.2%, re- presented in a later paragraph. spectively). In three experiments, the yields of ␣E-DC and Markers were sought to distinguish the two I-Ahigh DC popu- CD11bhigh-DC were 1.9 Ϯ 1.2 ϫ 105 and 1.7 Ϯ 0.5 ϫ 105 cells lations further. The CD11bhigh population characterized in earlier per mouse lung, respectively. Macrophages were recovered at studies (28) was found to constitute the major I-Ahigh DC popu- 5.4 Ϯ 3.9 ϫ 105 cells per lung. In normal lungs, the three major lation that expresses slightly higher I-A and lower CD11c (popu- ϩ CD11c populations ␣E-DC, CD11bhigh-DC, and macrophages lation 3 in Fig. 1Ab, 11b population in B, b–d). Because lung comprised 14 Ϯ 6.7%, 12.5 Ϯ 1.4%, and 54 Ϯ 3.9%, respectively, of the total CD11cϩ cells. Thus ␣E-DC is a major lung DC population, comprising 40–60% of the lung CD11cϩI-Ahigh DC. ␣E-DC express surface markers distinct from other DC types and mucosal intraepithelial lymphocytes DC from lymph node and spleen are classified into at least five populations according to their surface expression of CD4, CD8, DEC- 205, CD11b, and Langerin (16, 30). In addition, a CD11c-, B220-, GR-1-, and mPDCA-1-expressing PDC population (18–20) and an intestinal ␣E-DC (37) have been described. In lungs, CD4ϩ and CD8ϩ DC in the I-Aϩ and CD11cϩ fraction were not detected (Fig. 2B, b and c). Lung CD11cϩ DC expression of DEC-205 is also weak (data not shown). PDC yield in the anti-CD11c-magnetic bead-enriched fraction was variable, presumably due to the lower CD11c expression on its surface. However, PDC was readily isolated by anti- mPDCA-1-magnetic beads. Thus among the characterized DC subsets, only CD11bhigh-DC, ␣E-DC, and PDC were found in lungs. ␣E-DC is clearly distinct phenotypically from PDC. They express few of the PDC surface markers B220 and Gr-1 (Fig. 2A, b and c). In contrast, PDC express high levels of B220 and Gr-1 on their surface (Fig. 2A, e and f). In data not shown, mPDCA-1 was also absent on ␣E-DC. ␣E-DC do not express the lymphoid markers CD4 and CD8 expressed by some DC subsets in lymphoid tissues (Fig. 2B, b and c). It is also distinct from the mucosal intraepithelial lymphocytes, ␣ ␤ which are found in lungs and also express the E 7 integrin. As ␣ ␣ ␤ stated earlier, E-DC express the integrin E 7 (Fig. 2C) but not lymphocyte Peyer’s patch high endothelial venule adhesion mol- ␣ ␤ ecule 1 ( 4 7), whereas intraepithelial lymphocytes express both. ␣E-DC do not express CD3, TCR-␣␤, and TCR-␥␦ (Fig. 2Ba; FIGURE 1. Identification of I-AhighCD11chigh DC populations in lung. TCR expression not shown). The intraepithelial lymphocyte num- Anti-CD11c-magnetic microbead-purified lung cells were stained as de- bers in lung cell suspensions were analyzed (Fig. 2D). Lung di- ϩ scribed in Materials and Methods and analyzed. A, Two major populations gests contained 6.5% lymphocytes, 25% of which were CD3 T of I-AhighCD11cϩ populations (b, populations 2 and 3) were found in cells (6 ϫ 105 CD3ϩ cells per lung). Approximately 16% of the CD11cϩ populations isolated from total lung digests of naive mice. The ϩ ␣ ␤ CD3 T cells expressed E 7 integrin (Fig. 2D, b and c). Intra- major population 1 consists of macrophages. B, Identification of the two epithelial lymphocytes expressed no CD11c (Fig. 2Dd) and low ␣ high ϩ major DC populations as E-DC and CD11b -DC. Isolated CD11c levels of I-A by only a small percentage of the population (Fig. lung cells were stained and analyzed for five-color fluorescence on a 2De), and thus are readily distinguishable from ␣E-DC. They were FACSVantage SE flow cytometer. The cells were gated on 7-aminoacti- ␣ nomycin D-negative live cells and CD11cϩ cells (a) and further analyzed present in lower numbers in lungs than E-DC. In absolute num- ϳ ϫ 4 for PE-anti-CD103 vs allophycocyanin-Cy7-anti-CD11b (b), PE-anti- bers, only 9.0 10 intraepithelial lymphocytes as compared 4 CD103 vs FITC-anti-I-A (c), and allophycocyanin-Cy7-anti-CD11b vs with 19 ϫ 10 ␣E-DC were found per mouse lung. Between the ϩ ϩ FITC-anti-I-A (d). A total of 10,000 events were collected. This analysis CD4 and CD8 lung intraepithelial lymphocyte subsets, more ϩ has been performed three times. CD8 intraepithelial lymphocytes were found and the ratio of ␣ ␤ ϩ 2164 INTEGRIN E 7 LUNG MUCOSAL DC

CD8ϩ/CD4ϩ intraepithelial lymphocyte was 1.4 (Fig. 2Dc). The absolute cell numbers were 5.5 ϫ 104 and 3.8 ϫ 104 per lung for CD8ϩ and CD4ϩ T cells, respectively. The CD8ϩ intraepithelial lymphocytes that make up 40% of all lung CD8ϩ T cells and 8.6% of CD3ϩ T cells likely represent the major CD8␣␣ intraepithelial lymphocyte population (39). Although TCR␥␦ϩ T cells were important in mucosal immunoregulation, few ␥␦ϩ T cells were found. They comprised only 2.8% of total CD3ϩ T cells, but 35% of ␥␦-T cells were ␣ ␤ ␣␤ϩ E 7 integrin positive. The remaining lung T cells were TCR . Adhesion molecules are highly expressed on ␣E-DC ␣ ␤ Other adhesion molecules besides integrin E 7 are required for the migration of ␣E-DC to lungs and for determining the preferential localization of these DC in the lung (40). To quantify the expression of these adhesion molecules by ﬂow cytometry, CD11cϩ DC were stained with either FITC-conjugated anti- CD103 or anti-I-A mAb and PE-conjugated anti-adhesion molecule mAb. ␣E-DC can be distinguished from CD11bhigh-DC by CD103 positivity (Fig. 3Ab) or lower I-A staining in ﬂow cytometry dot plots (Fig. 3, Ac and Be). ␣E-DC were found to express the ␣ ␣ ␣ ␤ integrin subunits 4, 5, v (CD51), and 3 (CD61) (Fig. 3B, Table I). These molecules form heterodimers that function in DC

FIGURE 2. Lung ␣E-DC is distinct from intraepithelial lymphocytes and other DC subsets. CD11cϩ cells were isolated from lung single cell suspensions from naive mice by magnetic cell sorting. The retained CD11cϩ cells and flow-through CD11cϪ cells were analyzed for DC subset marker expression. A–C, Cells were gated on 7-aminoactinomycin D-negative live cells and CD11cϩ cells. D, Stained CD11cϪ flow-through cells were gated on the lymphoid population in forward scatter vs side scatter dot plots, 7-aminoactinomycin D-negative live cells, and CD3ϩ cells for T cell staining. A total of 10,000 cells were collected for each analysis. A, ␣ ␣ ␤ ϩ ϩ E-DC is distinct from PDC. Lung integrin E 7 CD11c cells were negative for PDC markers B220 (b) and Gr-1 (c). For comparison, PDC were isolated from lung digests by anti-mPDCA-1-magnetic microbeads and stained by control mAb (d), B220 (e), and Gr-1 (f). Cells were gated ϩ FIGURE 3. Surface marker expression on ␣E-DC. CD11c lung cells on live cells and mPDCA-1ϩ cells. B, ␣E-DC (circled population) do not isolated by magnetic cell sorting were stained and analyzed as indicated in express CD4 and CD8 splenic DC markers. C, ␣E-DC is positive for both ␣ ␤ ϩ the dot plots. Cells were gated on 7-aminoactinomycin D-negative live integrin E and 7 staining. B and C, CD11c lung cells were stained and ϩ Ϫ cells and CD11c cells. The ␣E-DC populations were circled. Bd, the analyzed as in A. D, Lung intraepithelial lymphocytes are CD11c and ϩ Mac-3 macrophages were also circled. In I-A-stained cells (A, c and d; B, I-AϪ or I-Alow. CD11cϪ lung flow-through cells gated on CD3ϩ cells were e–h; and C, f–h), the cell populations with the lower I-A staining is ␣E- stained and analyzed as indicated. This experiment was repeated twice with DC, as shown in Ac. All analyses were from the same experiment, and four similar results. similar experiments have been performed. The mean fluorescence intensities for the Ag on the y-axis for ␣E-DC are shown in each panel. The Journal of Immunology 2165

Table I. Activation of lung ␣E-DC in mice with asthma-like diseasea

Relative Mean Fluorescence Intensity

Surface Ag Control OVA-immunized

B7–1 (CD80) 2.3 Ϯ 0.6 2.9 Ϯ 0.7 B7–2 (CD86) 5.7 Ϯ 1.4 16.1 ؎ 6.2 B7-H1 (PD-L1) 2.4 Ϯ 0.8 2.8 Ϯ 0.1 B7-DC (PD-L2) 1.7 Ϯ 1.1 3.9 ؎ 0.6 ICOS-L 3.8 Ϯ 0.4 2.0 Ϯ 0.8 CD40 1.6 Ϯ 0.3 32.4 ؎ 18 ICAM-1 (CD54) 62.4 Ϯ 13.4 216 Ϯ 102 CD49d 8.4 Ϯ 2.3 18.9 ؎ 9.8 CD49e 22.9 Ϯ 8.6 16 Ϯ 6.2 CD51 4.7 Ϯ 2.0 3.0 Ϯ 0.3 CD61 1.7 Ϯ 0.3 5.9 ؎ 1.1 I-A 110 Ϯ 9.3 273 Ϯ 162 TLR2 6.4 Ϯ 0.9 7.6 Ϯ 1.7

a Puriﬁed lung DC were stained by mAb against CD11c, CD103, and the indicated markers and gated on the CD11cϩCD103ϩ population for activation analysis. The results are the average of four experiments. Data were normalized against isotype control and expressed as mean Ϯ SD. Bold indicates statistical signiﬁcance ( p Ͻ 0.05).

migration into lungs or bind to extracellular matrix proteins such as laminin and collagen. ␣E-DC also express high levels of ICAM-1 (see Fig. 5Bf) and some Mac-3 (Fig. 3Bd). The latter binds to the macrophage galactose glycoconjugate-binding protein galectin-3 (Mac-2) (41).

␣E-DC expression of costimulation molecules and TLR DC Ag presentation functions are dependent on their expression of costimulation molecules, which are up-regulated by TLR stimulation. The expression of these critical effector molecules on ␣E-DC was examined by flow cytometry. Among a panel of costimulation molecules examined, ␣E-DC was found to express moderate levels of several B7 molecules including B7-1, B7-2, B7-H1, and B7-DC (Fig. 3C, a–d; Table I). They also expressed the accessory molecules CD40 and Ox40L, which are important for DC activation and costimulation (Fig. 3C, e and f). The expression of TLR2 and TLR4, which are responsible for DC responses to bacterial prod- FIGURE 4. Characterization of lung macrophages and PDC. A, Pulmo- ucts, was also examined. Interestingly, ␣E-DC expressed TLR2, nary macrophages were isolated by anti-F4/80-magnetic microbeads and ϩ which responds to lipoteichoic acid and peptidoglycan but not gated on 7-aminoactinomycin D-negative live cells and CD11c cells. The TLR4, which responds to LPS (Fig. 3C, g and h). cells were stained and analyzed as indicated. B, PDC were enriched by anti-mPDCA-1-magnetic microbeads, gated on 7-aminoactinomycin ϩ Lung macrophages express high levels of B7-H1 and Siglac-F D-negative live cells and mPDCA-1 cells (a), and analyzed for DC sub- ϩ set-specific markers or costimulation molecules. These isolated PDC were Lung macrophages constitute a major CD11c population (Fig. also positive for B220 and Gr-1 as shown in Fig. 2A, e and f. The mean 1B). Their distinguishing surface markers were sought to facilitate fluorescence intensities for the Ag on the y-axis for encircled macrophage ϩ the comparison of their functions with DC in CD11c lung cell and PDC populations are shown in the dot plots. Two similar analyses with populations. In earlier experiments, lung macrophages were found the same results have been performed for each cell type. to express the splenic macrophage marker F4/80. Thus macrophages were enriched by anti-F4/80-magnetic beads from lung digests for more clear-cut marker studies (Fig. 4A). Greater than 3Bd, population M). Thus three specific markers, F4/80, B7-H1, 90% of the macrophages in the CD11cϩ fraction (population 1 in and mSiglac-F, are useful for lung macrophage identification. Be- Fig. 1Ab) were recovered by F4/80-magnetic microbead selection, cause B7-H1 is an inhibitory costimulation molecule (42), the high and both the selected and residual macrophages in the flow- expression of B7-H1 on lung macrophages may explain the inhib- through were found to express the same surface markers. A small itory function of pulmonary macrophages in T cell responses (45). percentage (16%) of these macrophages expressed low levels of I-A (compare Fig. 4A, a with b). Lung macrophages expressed Lung PDC express low levels of costimulation molecules high levels of CD11c (Fig. 4Ac). Besides expressing F4/80, they PDC may play a significant role in immune responses in lungs. The expressed high levels of two new surface markers, the inhibitory presence of PDC has been shown in lungs, but the DC population costimulation molecule B7-H1 (42) and the sialic acid-binding lec- has not been characterized (22, 28, 29). To examine lung PDC tin Siglac F, which is also highly expressed by eosinophils and surface marker expression, these cells were first enriched with anti- immature bone marrow macrophages (Fig. 2, d–f) (43, 44). A sub- mPDCA-1-magnetic microbeads followed by staining with anti- population of these macrophages was also positive for Mac-3 (Fig. I-A and other surface markers (Fig. 4B). In three experiments, ␣ ␤ ϩ 2166 INTEGRIN E 7 LUNG MUCOSAL DC

6.2 Ϯ 2.6 ϫ 104 PDC were isolated per lung. Besides PDC, the DC pinocytosed avidly with an initial burst of uptake in the first 20 anti-mPDCA-1 mAb also stained some other unspecified lung cell min (Fig. 5, Ab and D). This higher initial uptake rate was not seen populations (Fig. 4Bc). However, PDC can be identified readily as in CD11bhigh-DC, which pinocytosed at a linear but faster rate for a discrete population with low autofluorescence, intermediate I-A ϳ40 min before starting to reach plateau (Fig. 5D). The rapid expression (Fig. 2, Ad and Fig. 4, Bb), and high levels of B220 and FITC-dextran uptake by ␣E-DC was not due to mannose receptor- Gr-1 expression (Fig. 2A, e and f). PDC expressed no ␣E (Fig. mediated uptake because a 10-fold excess of S. cerevisiae mannan 4Bd), but low to intermediate levels of CD11b (Fig. 4Be). No failed to reduce FITC-dextran uptake rate appreciably (Fig. 5, Ac detectable amounts of B7-1 and CD40, and low levels of B7-2 and and D). The lack of mannose receptor involvement was also true B7-DC were found on their surface (Fig. 4B, f–i). Thus substantial for FITC-dextran uptake by CD11bhigh-DC and macrophages (Fig. numbers of PDC were found in mouse lungs, but they express low 5, Bc, Cc, and D). The finding was further supported by the failure of numbers of costimulation molecules. anti-mannose receptor (CD204) mAb to stain ␣E-DC (data not shown). Although ␣E-DC pinocytosed less rapidly compared with ␣ E-DC pinocytose avidly CD11bhigh-DC, their pinocytic rate was comparable to that of mac- An essential function of DC is Ag uptake. The pinocytosis of rophages, which are known to pinocytose rapidly (compare Fig. 5, A FITC-dextran by lung DC subsets was examined (Fig. 5). FITC- with B and C). The uptake of FITC-dextran was also readily observed dextran uptake by ␣E-DC was readily observed (Fig. 5Aa). These in lung DC and macrophages by confocal microscopy (Fig. 5E).

FIGURE 5. Pinocytosis of FITC-dextran by lung ␣E-DC, CD11bhigh-DC, and macrophages. CD11cϩ lung cells were incubated with FITC-dextran with (M) or without (C) mannan for the indicated time periods and stained with allophycocyanin-anti-IA plus PE-anti- CD103, CD11b, or Siglac-F. Live cells were gated on the indicated populations in I-A vs PE-conjugated subset-specific mAb plots and analyzed for FITC-fluorescence. The overlap of FITC-dextran fluorescence of the gated populations (A–C) at 0 (blue) and 60 min (red) are shown in a, the time course of FITC-dextran uptake is shown in b, and the competition of mannose receptor- mediated uptake by mannan at 0 and 60 min is shown in c. D, FITC-dextran uptake kinetics. ϩm represents plus mannan. E, A separate experiment showing FITC-dextran uptake by ␣E-DC, CD11bhigh-DC, and macrophages at 60 min is shown. a, The white arrows show an ␣E-DC and the white triangle marks a putative CD11bhigh-DC. Bars show 5 ␮m. Similar pinocytosis experiments have been performed three times. The Journal of Immunology 2167

␣E-DC stimulate T cell proliferation efficiently ␣E-DC produce IL-12 The potency of ␣E-DC in stimulating T cell proliferation was ex- Lymph node DC subsets have been shown to have different cyto- amined by using purified lung DC populations sorted by flow cy- kine production profiles (46). Lung ␣E-DC and CD11bhigh-DC tometry. The sorted populations were 98% pure and showed no production of the signature cytokines IL-12, IFN-␣, and IFN-␥ for overlap in marker staining between the two DC populations (Fig. different DC subsets were measured. Because flow cytometry anal- 6A). Both ␣E-DC and CD11bhigh-DC stimulated DO11.10 T cell ysis showed that lung DC expressed TLR2 (Fig. 3Cg) and that proliferation efficiently when either soluble anti-CD3 mAb or efficient DC stimulation required the activation by multiple TLR ligands (47), highly purified DC populations (Fig. 6A) were incu- OVA323–338 peptide was used as a stimulant (Fig. 6B). The stimulation potencies of ␣E-DC and CD11bhigh-DC were comparable, bated with combinations of ligands of different TLR or anti-CD40 mAb with the TLR2 ligand PAM CSK No IFN-␣ or IFN-␥ were and they were at least 20-fold more potent than irradiated spleno- 3 4. detected in DC culture supernatants. However, IL-12 production cytes on a per cell basis. was readily detected. Control cultures with no stimulants produced low levels of IL-12 (Fig. 6C). There were significant differences in responses between ␣E-DC and CD11bhigh-DC to the stimulants.

PAM3CSK4 combined with poly(I:C) were the strongest stimulants for ␣E-DC but had only moderate effects on CD11bhigh-DC.

PAM3CSK4 plus LPS, CpG, or anti-CD40 mAb, in contrast, had comparable effects on the two DC populations in IL-12 induction. Thus lung ␣E-DC had a higher response to TLR3 ligands than CD11bhigh-DC and both DC types are similar to the CD8ϩCD4Ϫ splenic DC subset in their ability to produce IL-12 (46).

␣E-DC are localized in the airway mucosa and perivascular region ␣E-DC are distinguishable from intraepithelial lymphocytes by their CD11c and I-A expression (Fig. 2D, d and e). Although eo- ␤ ϩ Ϫ sinophils are 7 integrin , they are CD11c-low and I-A (data not ␣ ␤ shown). Thus colocalization of the combinations of E or 7 integrin with either CD11c or I-A staining constitutes a reliable cri- terion for identifying ␣E-DC. Confocal microscopy of lung tissues ␣ ␤ stained with combinations of mAb against E, 7, CD11c, or I-A showed that these markers were colocalized and that ␣E-DC were present in large numbers mainly in the airway mucosa or on the parenchyma side of the arteriole walls (Fig. 7A). In the mucosa, ␣E-DC were found tightly apposed to the basal surface of bron- ␣ ␤ chial epithelial cells where the E 7 integrin ligand E-cadherin is present. DC were rarely seen within the bronchiolar epithelial layer (Fig. 7A). The intraepithelial network seen in airway tangential sections (10) likely represents the pseudopod extensions rather than the cell bodies of DC. The periarteriole ␣E-DC were found directly underneath the vascular endothelial cells, perhaps attached to the basal lamina (Fig. 7, A, d–f, and B, d–f). ␣E-DC constituted 70–75% of the I-AϩCD11cϩ-DC in the proximal subepithelial and periarterial regions. Unlike PDC, ␣E-DC were rarely observed in the alveolar septa. Some CD11bhigh-DC characterized by bright CD11b staining and coinciding with intense I-A and positive CD11c staining were also found in the perivascular regions (Fig. 7C), but few in the epithelial regions, of lung airways. Some single CD11bϩ cells representing neutrophils or eosinophils were also present in the mucosal as well as the alveolar regions (Fig. 7Cc, arrow).

␣E-DC expresses the epidermal DC marker Langerin To support the epithelial nature of ␣E-DC, an Affymetrix microarray database obtained from mRNA of sorted ␣E-DC and FIGURE 6. Ag presentation and IL-12 production by FACS-sorted pure CD11bhigh-DC was constructed and used to search for differen- ␣ high ␣ high E-DC and CD11b -DC populations. Sorted E-DC or CD11b -DC tially expressed genes. The mRNA of Langerin, an epidermal populations (A, 98% pure) were used to stimulated puriﬁed CD4ϩ splenic Langerhans cell marker shown to participate in Birbeck granule T cells from DO11.10 transgenic mice with either anti-CD3 (Ba)or OVA peptide (Bb) according to Materials and Methods and com- formation and nonpeptide Ag presentation (48, 49), was highly 323–338 ␣ ␣ pared with stimulation by irradiated splenocytes. The production of IL-12 expressed in E-DC, and its expression is 12-fold higher in E-DC high by the DC subsets after 36 h of stimulation with the indicated TLR ligands than in CD11b -DC (Table II). The microarray data were con- or anti-CD40 mAb is shown in C. A representative experiment from three ﬁrmed by real-time PCR, which also showed that ␣E-DC expressed similar experiments is shown. much higher (23-fold) Langerin mRNA than CD11bhigh-DC (Table ␣ ␤ ϩ 2168 INTEGRIN E 7 LUNG MUCOSAL DC

FIGURE 7. Confocal microscopy of ␣E-DC and CD11bhigh-DC in lungs. Lung tissues were prepared and sectioned as described in Materials and Methods. A–C, The indicated mAb directly conjugated with FITC (green label), PE (red label), or allophycocyanin (blue label) were used to stain the sections. Panels show single color, overlap of the three colors as indicated (merge), or further merging with phase-contrast images to locate the stained cells (merge-phase). Tissues in A and C, a–c, were from naive mice and B and C, d–f, were from mice immunized with OVA. Insets in b and e show CD11c staining (blue) of the bracketed area in the merged images. Single-positive cells in Cc are indicated by arrows. A magniﬁed image of the framed area in high ␤ Cf is shown in the inset. Arrows show CD11b -DC (yellow to white image). D and E, Anti-I-A and anti- 7 mAb were directly conjugated with ﬂuorophores, whereas Alexa dye 647-conjugated goat anti-rabbit IgG were used as secondary Ab against anti-Langerin and anti-Claudin-1 Ab. The staining of single color (Alexa dye 647, blue, a), overlap of two colors (b, green and red), and merged images of all three colors with phase-contrast images (c) are shown. Arrows indicate Langerinϩ (D) or Claudin-1ϩ (E) cells. Photomicrographs were captured with a ϫ40 objective and bars show 20 or 50 ␮m as indicated. ar, Arteriole; br, bronchiole; and epi, bronchiolar epithelium. These experiments have been repeated four times with different tissues from different experiments.

II). The Langerin mRNA product with 40 cycles of amplifications ray data showed that among the known tight junction proteins, migrated with the expected mobility (Fig. 8, lanes 3 and 4). Stain- Claudin-1, Claudin-7, and ZO-2 mRNA were overexpressed in ing of lung tissues by anti-Langerin Ab showed that Langerin ␣E-DC compared with CD11bhigh-DC (Table II). Further exami- staining was only colocalized with ␣E-DC, which showed bright nation of mRNA levels of the major tight junction proteins zona ␤ I-A and integrin 7 staining and are localized mostly either in the occludins (tight junction proteins), Claudins, and junction adhe- epithelium or arteriolar wall (Fig. 7D). The results confirmed that sion molecules showed that except ZO-2, Claudin-1, and Clau- ␣E-DC expressed Langerin and that it was the predominant cell din-7, ␣E-DC and CD11bhigh-DC expressed none or very low lev- type in lungs for this expression. The Langerin expression of els of the mRNA for these groups of proteins in the microarray ␣E-DC suggests that they are similar to Langerhans cells, CD8ϩ data set (data not shown). Real-time PCR confirmed that ␣E-DC lymphoid DC, and DEC-205highCD8low lymph node DC, all of expressed much higher levels of Claudin-1 and Claudin-7 and which express this protein (50, 51). somewhat higher level of ZO-2 than CD11bhigh-DC (Table II). The PCR products exhibited the expected migration mobilities (Fig. 8, ␣ E-DC express Claudin-1, Caludin-7, and ZO-2 lanes 5–10). Staining of lung tissues for Claudin-1 and Claudin-7 To gain access to Ag or pathogens in the airways, ␣E-DC in the showed that these tight junction proteins are expressed by ␣E-DC mucosa must traverse the bronchial epithelial layer through the (Claudin-1, Fig. 7E; Claudin-7 is specifically expressed in ␣E-DC tight junction barrier. This is achieved by the ␣E-DC surface ex- but is dim and, therefore, not shown) and the staining is colocal- ␤ pression of tight junction proteins, which interact with those on the ized with I-A and 7 in the epithelia or vascular walls. These epithelial cells and allow the cell body to squeeze through (re- results showed that ␣E-DC are equipped with tight junction pro- viewed in Ref. 52). Differential discovery of Affymetrix microar- teins that will enable them to migrate or extend their pseudopods The Journal of Immunology 2169

Table II. Expression of Langerin and tight junction protein mRNA by lung DCa

Microarray

␣E_DC CD11b_DC ␣E_DC CD11b_DC

Affymetrics Accession Mean (SD)b Mean (SD) Name I.D. No. Mean (SD) Mean (SD) ␣E/CD11b ratio ϫ 103 ratio ϫ 103 ␣E/D11b

CD207 (Langerin) 1425243 AY026050 2419 (476) 202 (69) 12.00 16.05 (1.72) 0.71 (0.49) 22.71 Claudin_1 1437932 AV227581 1575 (121) 199 (126) 7.88 13.48 (1.98) 2.66 (0.10) 5.06 1438851 BB210412 1282 (90) 229 (125) 5.58 1450014 NM_016674 1525 (494) 160 (72) 9.50 Claudin_7 1448393 BC008104 208 (27) 25 (4) 8.00 0.85 (0.06) None detected —c ZO_2 1434599 BB758095 153 (9) 68 (8) 2.22 0.83 (0.03) 0.72 (0.18) 1.15 1434600 BB758095 154 (3) 69 (4) 2.24

a mRNA expression of the indicated genes in ␣E-DC or CD11bhigh-DC were determined by Affymetrix microarray or real-time PCR as described in Materials and Methods. The mean of three independent determination and standard deviations are shown for microarrays, and one representative experiment of two experiments is shown for real-time PCR. The signal ratios of mRNA in ␣E-DC vs that in CD11bhigh-DC are shown. b The ratio of calculated mRNA value of unknown normalized against that of ␤-actin is multiplied by 1000. c The ratio of mRNA in ␣E-DC vs that in CD11bhigh DC cannot be determined. Formally it is ϱ.

into the bronchiolar luminal space to capture Ag for processing terestingly, the preferential localization of ␣E-DC and and presentation. CD11bhigh-DC were different. ␣E-DC remained largely immediately adjacent to the basal lamina of the bronchial epithelia and ␣ ϩ ϩ Lung E-DC accumulation and activation in mice with Ag- arterioles, where 65–70% of the I-A CD11c -DC were integrin induced inflammation ␣ ␤ ϩ high E 7 (Fig. 7B). CD11b -DC, in contrast, were found in the ϩ ϩ The role of lung ␣E-DC in asthma was examined in an OVA- proximal subepithelia and vascular wall as a minor I-A CD11c induced asthma-like model (33). Immunized mice exhibited population (Fig. 7C, e and f). They occurred primarily interspersed marked airway hyperresponsiveness and eosinophilia. ␣E-DC in- within the leukocyte infiltration zone of the peribronchial and creased markedly, from an average of 1.9 ϫ 105 in control mice to perivascular cuffs (Fig. 7Cf, inset, arrows). The results suggest that 5.4 ϫ 105 ␣E-DC per lung in immunized mice. They constituted ␣E-DC and CD11bhigh-DC exhibit different preferential 21% of the total lung I-AhighCD11chigh-DC. The increase was not localization. due to residual injected DC in lungs based on three lines of evidence. The first is that CFSE-labeled bone marrow-derived DC injected into lungs disappear rapidly and no residual fluorescence Discussion was found by day 7, when the label was found exclusively asso- Although DC are considered the critical cell type for airway im- ciated with macrophages in the paracortical region of thoracic munity (3–6), lung DC subsets are poorly characterized. With the lymph node, likely representing residual fluorescent material from combination of magnetic microbead sorting followed by FACS phagocytosed exogenous DC. The second is that when 1 ϫ 106 purification, we obtained 5 ϫ 103 each of 98% pure ␣E-DC and CD45.2 bone-marrow-derived DC were injected intratracheally CD11bhigh-DC (1 ϫ 104 DC per mouse total), a 4-fold increase in into CD45.1 mice, exogenous DC constituted Ͻ1% of the total yield regarding I-Ahigh lung DC when compared with previous ϩ CD11c lung cells in unchallenged mice on day 6. These injected reports (23, 27, 28). Besides myeloid DC, PDC have been de- cells expressed no ␣E. In OVA-immunized mice, ␣E-negative scribed in lung cell digests, but the numbers are too small for lung DC increased by at least 12-fold compared with controls. phenotypic and functional analyses (22, 28). Furthermore, von Thus exogenous DC constituted at the most 0.7% of the ␣E-neg- Garnier et al. have detected PDC in their CD11clow and CD11cϪ ative DC. This number is most likely to be much lower because of lung cell fractions that are not normally analyzed for DC occur- cellular activation, mobilization, and apoptosis in the inflamed rence, and PDC numbers are small in the CD11cϩ lung cell pop- lung. The third line of evidence is that, in the study by van Rijt et ulation (29). The yield of PDC by anti-CD11c-magnetic microbead al. (6) using a similar immunization protocol, no residual exoge- isolation in our hands was similarly variable. Using magnetic nous DC was detected. ␣E-DC in asthma-induced mice were ac- beads specific for PDC, we were able to enrich for PDC in lung tivated, with significant increases in surface expression of B7-2, digests. The yield was estimated to be 6 ϫ 104 PDC per lung. Thus B7-DC, CD40, and CD49d expression (Table I). ICAM-1 and PDC occurrence is approximately one-third that of either ␣E-DC MHC class II expression also showed consistent increases in in- or CD11bhigh-DC in lungs. CD8ϩ and CD4ϩ lymphoid DC have tensities on ␣E-DC. However, the increase did not reach statistical also not been identified in lungs. Our results showed that few if any significance because of the high scattering of the results from rep- of these lymphoid DC were present in control (Fig. 2B) and in- licate experiments. flamed lungs (data not shown). It is noteworthy that the migrating airway DC that have captured intratracheally introduced FITC- ␣ high Large numbers of E-DC and CD11b -DC were present in OVA were reported to be CD8ϩ (13). The results suggest that the the mucosa of inflamed lungs CD8 marker is induced in lung DC upon activation and migration Confocal microscopy showed that as in control lungs, large num- to the lymph node. Epidermal Langerhans cells have been shown bers of ␣E-DC and CD11bhigh-DC were present in the airway mu- to express CD8 similarly at a low to intermediate level (16). Thus cosa, adjacent to the vascular walls, and within the leukocyte in- in lungs, three major I-Aϩ DC types have been identified in this filtration areas (Fig. 7B and 7C, d–f). Many more CD11bhigh-DC study. In addition to these three subsets, a mucosal CD11bhigh-DC were found (Fig. 7C), in agreement with the increases in lung population in the respiratory tract with high turnover rate has also CD11bhigh-DC numbers (12-fold higher) in immunized mice. In- been identified (29). ␣ ␤ ϩ 2170 INTEGRIN E 7 LUNG MUCOSAL DC

data has been reported when splenic CD8ϩ DC mRNA expression was compared with that of either CD4ϩ or double-negative splenic myeloid DC (54). The differential overexpression of Notch 4 mRNA by ␣E-DC is of considerable interest because of the regulation of Th1 and Th2 responses by Notch signaling in T cells FIGURE 8. Amplified Langerin and tight junction protein cDNA prod- (55) and the relevance of Th2 cells in asthma. Of the four Notch ucts migrated with the expected mobilities. cDNA transcribed from total ␣ high genes, detectable mRNA expression in freshly isolated lung RNA of sorted E-DC (lanes a) or CD11b -DC (lanes b) were amplified ϩ for 40 cycles as described in Materials and Methods. PCR products were CD11c DC was observed only for Notch 1 and Notch 4 by mi- fractionated in 1.8% agarose gels. The expected product sizes were: CD207 croarray analysis. The signal intensities for Notch 1 mRNA were high (Langerin), 178 bp; Claudin-1, 151 bp; Claudin-7, 213 bp; ZO-2, 171 bp; 106 Ϯ 7 and 197 Ϯ 59 for ␣E-DC and CD11b -DC, respec- and ␤-actin, 181 bp. Lanes 1 and 2 contained no cDNA. This analysis was tively, and the corresponding values for Notch 4 mRNA were performed twice. 629 Ϯ 93 and 39 Ϯ 3 (S. J. Sung, unpublished results). Notch signaling has been shown to induce the differentiation of hemo- poietic precursors into myeloid DC precursors (56) and the mat- ␣E-DC have not been described in lungs previously; however, uration of human monocyte-derived DC (57). However, the sig- they have been found in the mesenteric and other lymph nodes (37) nificance of the Notch 4 expression by ␣E-DC in modulating lung and in rat small intestine lamina propria (32, 38). In the skin- immune response has yet to be determined. The expression of ␣ ␤ draining lymph node, integrin E 7-expressing DC have also been mRNA for the Th subset-directing Notch receptor ligands Jagged isolated (17). However, these DC are postulated to have acquired 1, Jagged 2, and Delta 1 by these lung DC in microarrays were also ␣ ␤ their E 7 integrin in the draining lymph node and thus may be examined. Although little Delta 1 mRNA was observed, detectable different from those found in the lung mucosa. Lung ␣E-DC are levels of Jagged 1 (75 Ϯ 25, 148 Ϯ 57) and Jagged 2 (82 Ϯ 20, clearly distinct from intraepithelial lymphocytes by their lack of 41 Ϯ 9) mRNA were observed in ␣E-DC and CD11bhigh-DC, CD3, TCR␣␤, TCR␥␦, CD4, or CD8 expression (Fig. 2B), and by respectively. It is likely that the mRNA and protein expression of the absence of CD11c and I-A on intraepithelial lymphocytes (Fig. these Notch receptor ligands are induced upon lung DC activation. 2D, d and e). They are two to three times more numerous than It will be important to determine whether DC Notch ligand ex- intraepithelial lymphocytes in the lung and may be in even higher pression and stimulation is a key mechanism for directing Th2 percentages in inflamed lungs. In the lung epithelia and arteriolar responses in asthma. ␣ ϩ ␤ ϩ ϩ ␣ walls, most integrin E or 7 cells were I-A DC (Fig. 7). The The difference in cellular properties between E-DC and majority of the intraepithelial lymphocytes in lungs (60%) were CD11bhigh-DC suggests that these two DC subsets may serve dif- the CD8ϩ T cells, which likely represent the CD8␣␣ϩTCR␣␤ϩ ferent functions in lung inflammation. ␣E-DC are tightly apposed intraepithelial lymphocytes. These cells were regulatory in func- to the basolateral side of bronchial epithelial cells where E-cad- ␣ ␤ tion and may have relevance in asthma pathogenesis (53). herin, the ligand for E 7, occurs (Fig. 7). Coupled with their In this report, we have identified the mucosal ␣E-DC as a major surface expression of tight junction proteins such as Claudin-1, DC population in mouse lungs. They are distinct from the previ- Caludin-7, and ZO-2, ␣E-DC can open up tight junctions between ously described major lung myeloid CD11bhigh-DC population epithelial cells and either send their dendrites or directly migrate (28) with regard to surface marker expression. Compared with across the bronchial epithelium into the airway for Ag capture, as ␣ high ␣ ␤ ␣ E-DC, CD11b -DC express no E 7, higher levels of I-A, have been demonstrated for intestinal DC (58). Because E-DC much higher levels of CD11b, and lower level of CD11c. In in- constitutes 75% of the DC population immediately adjacent to the flamed lungs, stimulated CD11bhigh-DC expressed much higher epithelia, they are likely to be the DC population observed to form levels of B7-H1 and B7-DC than ␣E-DC (data not shown). There an I-Aϩ reticular network in the airway mucosa (10). In inflamed were also functional differences between these two DC subsets. lungs, ␣E-DC remained the main DC population in the epithelium, ␣E-DC pinocytosed at a lower rate, but with an initial burst that is despite a much larger increase in lung CD11bhigh-DC. These ob- absent with CD11bhigh-DC (Fig. 5). Furthermore, ␣E-DC re- servations suggest that a major function of ␣E-DC may be to cap- sponded to TLR3 stimulation with a higher production of IL-12 ture Ag in the airways and transport the Ag to the thoracic lymph (Fig. 6C). In terms of tissue occurrence, ␣E-DC are preferentially node for T cell priming. CD11bhigh-DC, in contrast, are found localized at the basal lamina of the bronchial epithelia and arte- interacting with other leukocyte cell types in the peribronchial and rioles (Fig. 7, A and B), whereas most of the CD11bhigh-DC are perivascular leukocyte infiltrating areas. They may be responsible present more distal to the basal lamina, and are within the leuko- for activating infiltrating leukocytes. CD11bhigh-DC may also be cyte infiltrating area in inflamed lungs (Fig. 7C). ␣E-DC also ex- important for leukocyte recruitment. DC have been shown to pro- press Langerin and markedly higher levels of tight junction produce large amounts of CC and CXC chemokines such as thymus- teins. These results support the hypothesis that ␣E-DC and and activation-regulated chemokine, macrophage-derived chemo- CD11bhigh-DC are distinct DC populations. kine, and IFN-inducible protein 10 (59). The interaction of Several lines of evidence suggest that ␣E-DC exhibit pheno- CD11bhigh-DC with other inflammatory leukocytes may induce typic and functional characteristics similar to that of lymphoid DC. high levels of chemokines for further leukocyte recruitment. Thus The first is that ␣E-DC express Langerin. Langerin mRNA has there may a dichotomy of functions for ␣E-DC and CD11bhigh- been detected in the CD8highCD11blow population of spleen and DC, the former being responsible for airway Ag transport to the LN cells (51). Secondly, among CD11chigh splenic DC, integrin draining lymph node and the latter for propagating the local lung ␣E mRNA was expressed only in CD8␣ϩ, but not in CD4ϩ or inflammatory response. If the hypothesis is correct, then ␣E-DC double-negative DC subsets (54). The third is that in microarray will be more mobile while CD11bhigh-DC will preferentially re- analyses, several genes have been found to be expressed at much main in lungs during Ag challenges. higher levels in ␣E-DC than in CD11bhigh DC. These genes in- Although ␣E-DC may play a key role in capturing Ag in the clude CD24a (14-fold), Notch 4 (11-fold), and IFN consensus se- airway, they may also be important in capturing pathogenic Ag in quence binding protein 1 (8-fold) (S. J. Sung, unpublished results). the interstitium and in interacting with infiltrating leukocytes in the Similar overexpression of these genes by CD8ϩ DC in gene chip perivascular and peribronchial cuffs. The finding that primed T The Journal of Immunology 2171 cells interact with DC in the epithelium suggests that T cell-DC and presentation, produce IL-12 upon TLR stimulation, express interaction in the lungs is a significant and common event (14, 15). tight junction proteins that allow them to traverse the bronchial However, the full extent of the consequences of these interactions epithelium readily, and increase markedly in number during lung in lungs is unknown. Because of the rapid turnover of lung DC antigenic challenges. Studying the migration of this DC type dur- (10), there will be large numbers of DC in transit between the ing antigenic challenge and the functional role of this cell type in vasculature and the mucosa and between the mucosa and the lym- lung inflammation will likely provide mechanistic insight on the phatic ducts at any instant for Ag or cellular encounters. Further- pathogenesis of lung diseases. more, the trophic properties of Ag and pathogens leading to the release of chemotactic factors such as f-Met-Leu-Phe, complement Acknowledgments fragments such as C5a, and TLR ligands will induce the migration We thank Wei Shan, Elise Hackett, Binru Wang, Runpei Wu, of DC toward the attractant source. In addition to the mucosal Waunema Smith, and Natalie Walker for technical assistance, Hao M. Qian ␣E-DC, many ␣E-DC are also present on the parenchymal side of for advice, and A. Melissa Loggans for manuscript preparation. the pulmonary arterioles with ready access to the interstitium (Fig. 7, A and B). The exact significance of this vascular localization for Disclosures ␣ E-DC is unclear. However, an earlier report has also shown that The authors have no financial conflict of interest. DC are enriched on the luminal side of the vascular wall (60). These blood vessel-associated DC may serve in recruitment of References leukocytes into the lung perivascular region by the production of 1. Steinman, R. M. 2003. Some interfaces of dendritic cell biology. APMIS 111: chemokines. 675–697. Besides bearing DC hallmark surface Ag such as CD11c and 2. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of ␣ immunity. Nature 392: 245–252. MHC class II molecules, E-DC are proficient in performing the 3. Holt, P. G., and J. W. Upham. 2004. The role of dendritic cells in asthma. 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