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The Journal of Immunology, 2006, 176: 2161-2172.
Copyright © 2006 by The American Association of Immunologists

A Major Lung CD103 ({alpha}E)-beta7 Integrin-Positive Epithelial Dendritic Cell Population Expressing Langerin and Tight Junction Proteins1

Sun-Sang J. Sung2,*,{ddagger}, Shu Man Fu*,{ddagger}, C. Edward Rose, Jr.*, Felicia Gaskin{ddagger},{dagger}, Shyr-Te Ju*,{ddagger} and Steven R. Beaty*,{ddagger}

* Department of Internal Medicine and {dagger} Department of Psychiatric Medicine, and {ddagger} University of Virginia Specialized Center of Research in Systemic Lupus Erythematosus, University of Virginia School of Medicine, Charlottesville, VA 22908


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 organs. The third and largest DC population is the integrin {alpha}E (CD103) beta7-positive and I-AhighCD11chigh-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 {alpha}Ebeta7+ DC ({alpha}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, {alpha}E-DC numbers were increased in lungs, and their costimulation and adhesion molecules were up-regulated. These studies show that {alpha}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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Dendritic cells (DC)3 are the predominant APC type (1, 2) and play critical roles in airway antigenic and pathogenic responses (3, 4, 5, 6). DC processes extend into the epithelia to form an I-A+ reticular structure for Ag capture (7, 8, 9). In the homeostatic state, DC turnover occurs rapidly in the airway epithelium with a 2-day half-life (10). Enhanced lung DC migration to the draining lymph nodes is initiated by TLR ligands or Ag-specific T cells (11, 12, 13, 14, 15) with the appearance of Ag-loaded DC in the thoracic lymph node within 6 h and peaking between 2 and 3 days (11, 12, 13).

The major DC hallmark is their potent Ag presentation capability. DC have been classified according to their surface marker phenotype and functions into myeloid, lymphoid, and plasmacytoid DC (PDC) (1). In mice, five populations of lymph node DC have been described (16). Among them are the double-negative CD4–CD8–CD11b+ myeloid DC, the CD4+CD11b+ myeloid DC, and the CD8+ lymphoid DC present in both spleen and lymph node, plus two additional lymph node populations that are either CD8–DEC-205high or CD8lowDEC-205low. The DEC-205highCD8low population expresses Langerin, and is postulated to represent the matured form of Langerhans cells that has migrated to the lymph node. In that regard, a similar subset of CD11chighCD40highCD8{alpha}int DC population that also expresses {alpha}1beta1 and {alpha}Ebeta7 has been found in the skin-draining lymph node (17). Besides these conventional DC subsets with varying lineage or tissue origin, an IFN-{alpha}-producing PDC has been described in mice (18, 19). These cells are CD11c+I-A+B220+Gr-1+. A new marker described for PDC (20, 21) will facilitate the isolation and further characterization of PDC in lungs. Functionally, lung PDC has been shown to be important in suppressing antigenic responses in lungs (22). Lung DC isolated in mouse, rat, and human (23, 24, 25, 26) are MHC class II+ but mostly exhibit an immature phenotype. Mouse lung DC characterized thus far belong to the CD11bhigh myeloid population (27, 28, 29). No CD8{alpha}+ 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 GR-1 and B220 expression have been detected in the lung alveolar septa by immunohistochemistry (28). Recent studies of lung cells in excised lungs and respiratory tracts also showed that PDC is present in low numbers in both CD11c+ and CD11c– populations identified by a PDC-specific mAb, although these populations have not been examined in detail (29). In addition to the myeloid and PDC subset, a rapidly migrating CD11bhighCD11clowI-A– population has been found in the respiratory tract.

DC subsets have been shown to produce distinct cytokines, mediate different Th subset responses, present autoantigens through their ability to internalize apoptotic cells, regulate antigenic responses, and migrate differently in response to chemokines (1, 22, 31, 32). To understand the regulation of immune responses in the normal and diseased states in lungs, it is critical to characterize subset DC and study their functions separately. In this report, lung CD11c+I-Ahigh-DC populations enriched by anti-CD11c-magnetic microbeads have been resolved into two populations, an integrin {alpha}Ebeta7+ DC population ({alpha}E-DC) and a CD11bhigh population (CD11bhigh-DC), based on their integrin {alpha}Ebeta7, CD11b, I-A, and CD11c cell surface expression. Immunofluorescence microscopy showed that the {alpha}E-DC were mainly localized in the lung epithelia and in the arteriolar wall of naive and immunized mice. Flow cytometry analyses further showed that these DC constitute a major population of the I-AhighCD11chigh-DC in lungs. {alpha}E-DC are distinct from the CD11bhigh lung DC in surface phenotype, functional characteristics, and lung localization site, and they are clearly different from intraepithelial lymphocytes and PDC isolated by anti-mPDCA-1 magnetic microbeads. Functionally, {alpha}E-DC internalize FITC-dextran avidly, stimulate anti-CD3 and Ag-dependent T cell proliferation efficiently, and produce IL-12 upon stimulation by TLR ligands. In mice with induced asthma, lung {alpha}E-DC numbers and costimulation and adhesion molecule surface expression increased. Importantly, these DC express tight junction proteins Claudin-1, Claudin-7, and ZO-2 that will allow them to traverse the lung epithelia readily. Furthermore, {alpha}E-DC express Langerin, which suggest that they are similar to CD8+ lymphoid-derived DC and Langerhans cells. The results show for the first time that {alpha}E-DC constitute a major DC population residing in the lung mucosa, are competent in key DC functions, reside at specific mucosal locations, and exhibit an activated phenotype in asthma-induced mice. They may play key roles in airway antigenic responses and asthma.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Materials

TLR ligands were purchased from InvivoGen and have been tested to be LPS-free by the manufacturer. All mAb with and without fluorophore conjugation were purchased from eBioscience except the following: anti-CD4, integrin beta7, SiglacF, CD54, CD49e, and TCR mAb were obtained from BD Pharmingen; anti-F4/80 mAb was from Caltag Laboratories; anti-CD205 mAb was from Serotec; anti-mPDCA-1 mAb was from Miltenyi Biotec; anti-Claudin-1, Caludin-7, and ZO-2 rabbit polyclonal Ab were from Zymed Laboratories; and rabbit anti-Langerin Ab was from Imgenex. Alexa dye-conjugated secondary Ab were from Molecular Probes. ELISA kits for IFN-{gamma}, IL-12 p70, and IFN-{alpha} determination were from Endogen/Pierce, R&D Systems, and PBL Biomedical Labs, respectively. FITC-dextran and Saccharomyces cerevisiae mannan were purchased from Sigma-Aldrich.

DC purification

Lung single-cell suspensions were prepared essentially as described (33). Total lung DC preparations were obtained by CD11c-magnetic microbead selection according to the manufacturer’s protocol (Miltenyi Biotec). PDC were isolated from lung digests by magnetic cell sorting with anti-mPDCA-1 magnetic microbeads. Macrophages were isolated by anti-F4/80-magnetic microbeads. Pure {alpha}E-DC and CD11bhigh-DC populations were isolated from CD11c+ cells by sorting on a FACSVantage SE flow cytometer.

Mice and immunization

The immunization of BALB/cByJ mice for asthma induction was performed as described (33) with two notable exceptions: 1) animals were adoptively transferred i.v. with 2.5 x 106 splenic CD4+ DO11.10 transgenic T cells two days before DC sensitization; and 2) mice were sensitized intratracheally with bone marrow-derived DC (34) instead of splenic DC. The protocols in this study have been approved by the University of Virginia Institutional Use and Care of Animals Committee.

Flow cytometry analysis

Lung and lymph node cells were blocked with anti-Fc{gamma}RII/Fc{gamma}RIII mAb 2.4G2, stained with fluorophore-conjugated mAb, and analyzed by flow cytometry using FACSCalibur (BD Biosciences). Dead cells were excluded from analysis by 7-aminoactinomycin D staining. Flow cytometry results were analyzed by the program FlowJo (Tree Star).

Immunofluorescence and confocal microscopy

Lung tissues were fixed-inflated in 0.7% paraformaldehyde as described (33), equilibrated in 30% sucrose, and embedded in OCT. Sections (5 µm) were extracted with 0.3% Triton X-100, blocked with anti-Fc{gamma}RII/Fc{gamma}RIII mAb 2.4G2 and serum, and stained with primary and secondary Ab. Confocal microscopy was performed on a Zeiss LSM510 assembly with 488, 546, and 633 excitation lines. Data were compiled using the software provided by the manufacturer.

DC pinocytosis of FITC-dextran

Lung CD11c+ DC were suspended at 1 x 106 cells/ml, preincubated at 37°C for 15 min with and without 5 mg/ml mannan, and allowed to pinocytose 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, FITC-dextran uptake was measured by flow cytometry for I-AhighCD103+, I-AhighCD11bhigh, and I-A+Siglac-F+ cells representing {alpha}E-DC, CD11bhigh-DC, and macrophages, respectively.

DC stimulation of T cell proliferation

Spleen CD4+ T cells from DO11.10 transgenic mice were purified to 98% purity by depletion with magnetic microbeads conjugated with anti-CD19, CD11c, CD8, and DX5 mAb. Proliferation was performed as described (35). T cells were stimulated with sorted {alpha}E-DC or CD11bhigh-DC and either 2 µg/ml anti-CD3 mAb or 5 µM OVA323–339 peptide. Total splenocytes irradiated for 25 Gy were used as control APC.

Microarray analysis of {alpha}E-DC and CD11bhigh-DC mRNA

Magnetic bead-purified CD11c+ cells from lung digests were stained with anti-IA-FITC, anti-CD103-PE, anti-CD11c-allophycocyanin, anti-CD11b-Cy7-allophycocyanin, and 7-aminoactinomycin D and sorted for the I-AhighCD103+CD11c+CD11blow ({alpha}E-DC) and the I-AhighCD103–CD11c+CD11bhigh (CD11bhigh-DC) populations in the live cell gate in a two-way sort. Total RNA was extracted immediately with an RNeasy kit (Qiagen), and good quality RNA samples were obtained based on the profiles of 28S and 18S RNA fractionated on an Agilent Bioanalyzer and the 5' to 3' ratios of housekeeping genes in Affymetrix gene chip analysis. Gene expression levels in the two DC populations were probed with the Affymetrix Gene-Chip mouse genome 430 2.0 array by the University of Virginia Biomolecular Facility. Three independent pairs of {alpha}E-DC and CD11bhigh-DC from different isolations were analyzed by the same chip batch. The cell intensity files provided by the Affymetrix MAS program were further normalized and background was subtracted to provide gene expression levels using the program dChip (36).

Real-time PCR analysis of DC mRNA

Aliquots of total RNA from FACS-sorted DC subsets were reverse transcribed by the Advantage RT-for-PCR kit (BD Clontech). Real-time PCR was performed in a Bio-Rad iCycler Thermal Cycler using Sybr green fluorescence as the readout, and data were analyzed by the iCycler program provided (Bio-Rad). PCR conditions were as follows: 94°C for 22 s, 62°C for 30 s, and 72°C for 30 s for 39 cycles; 94°C for 22 s, 62°C for 30 s, and 72°C for 5 min for 1 cycle. Melt curves were obtained by increasing the temperature from 65°C to 95°C in 0.5°C increments for 10 s. The primer sequences were generated by the program Primer 1, and the primers were synthesized by IDT. The cDNA amplified, 5'-primer sequence, 3'-primer sequence, and product size, respectively, are as follows: Claudin-1, 5'-CCCAGTGGAAGATTTACTCCTAGT-3', 5'-TGCAAAGTACTGTTCAGATTCAGC-3', 151 bp; Claudin-7, 5'-GCTTCTTAGCCATGTTTGTCG-3', 5'-CAAACTCGTACTTAACGTTCATGG-3', 213 bp; ZO-2, 5'-CTGCTCAATTACACTCAGTGTTCT-3', 5'-GGCTGTAAAAAGATGAGAACAGGT-3', 171 bp; Langerin, 5'-ACAAGGAGCAAAGTAGGAGGTTCT-3', 5'-CAGATCTGTCATTCAGTTGTTTGG-3', 178 bp; and beta-actin, 5'-CTCTTTTCCAGCCTTCCTTCTTGG-3', 5'-CTCCTTCTGCATCCTGTCAGCAAT-3', 181 bp.

Statistics

The mean and SD of multiple trials were calculated by Excel (Microsoft). Statistical significance using Student’s t test was determined by the program SlideWrite plus (Advanced Graphics Software).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
{alpha}E-DC and CD11bhigh-DC are the two major populations of I-Ahigh lung DC

Previous studies have identified the CD11bhigh-DC as the only major DC populations in lungs (27, 28, 29). However, when anti-CD11c-magnetic bead-enriched DC from lung digests were analyzed by flow cytometry, two different I-Ahigh DC populations based on I-A and CD11c expression levels were found (Fig. 1Ab, populations 2 and 3). The mean fluorescence intensities for populations 2 and 3 were: I-A, 217 and 388, and CD11c, 153 and 22, respectively. A third major population comprising ~50% of the CD11c+ cells and with high autofluorescence and CD11c staining (Fig. 1, Ab, population 1, and B, b–d, population M) are pulmonary macrophages, the further characterization of which will be presented in a later paragraph.


Figure 1
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  		FIGURE 1. Identification of I-AhighCD11chigh DC populations in lung. Anti-CD11c-magnetic microbead-purified lung cells were stained as described in Materials and Methods and analyzed. A, Two major populations of I-AhighCD11c+ populations (b, populations 2 and 3) were found in CD11c+ populations isolated from total lung digests of naive mice. The major population 1 consists of macrophages. B, Identification of the two major DC populations as {alpha}E-DC and CD11bhigh-DC. Isolated CD11c+ lung cells were stained and analyzed for five-color fluorescence on a FACSVantage SE flow cytometer. The cells were gated on 7-aminoactinomycin D-negative live cells and CD11c+ cells (a) and further analyzed for PE-anti-CD103 vs allophycocyanin-Cy7-anti-CD11b (b), PE-anti-CD103 vs FITC-anti-I-A (c), and allophycocyanin-Cy7-anti-CD11b vs FITC-anti-I-A (d). A total of 10,000 events were collected. This analysis has been performed three times.

 
Markers were sought to distinguish the two I-Ahigh DC populations further. The CD11bhigh population characterized in earlier studies (28) was found to constitute the major I-Ahigh DC population that expresses slightly higher I-A and lower CD11c (population 3 in Fig. 1Ab, 11b population in B, b–d). Because lung contains an extensive epithelial surface, it is likely that the {alpha}E-DC found in other epithelial tissue such as rat intestinal mucosa (37, 38) would constitute a significant lung DC population or perhaps population two of the I-Ahigh DC (Fig. 1Ab). This hypothesis was found to be indeed the case. The lung I-Ahigh population with slightly higher CD11c but lower I-A expression (population 2 in Fig. 1Ab) was found to be integrin {alpha}E+ ({alpha}E population in Fig. 1B, b and c) but did not express lymphocyte Peyer’s patch high endothelial venule adhesion molecule 1 ({alpha}4beta7; data not shown). This population has a low to intermediate surface CD11b expression (Fig. 1Bb). However, a small population of these {alpha}E-DC ({approx}5% of {alpha}E+ cells) was CD11bhigh representing perhaps activated {alpha}E-DC.

Large numbers of the two I-Ahigh DC types could be isolated by magnetic bead sorting. {alpha}E-DC and CD11bhigh-DC each comprised ~1% of the total lung cell digests (0.7–1.6% and 1.1–1.2%, respectively). In three experiments, the yields of {alpha}E-DC and CD11bhigh-DC were 1.9 ± 1.2 x 105 and 1.7 ± 0.5 x 105 cells per mouse lung, respectively. Macrophages were recovered at 5.4 ± 3.9 x 105 cells per lung. In normal lungs, the three major CD11c+ populations {alpha}E-DC, CD11bhigh-DC, and macrophages comprised 14 ± 6.7%, 12.5 ± 1.4%, and 54 ± 3.9%, respectively, of the total CD11c+ cells. Thus {alpha}E-DC is a major lung DC population, comprising 40–60% of the lung CD11c+I-Ahigh DC.

{alpha}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, 19, 20) and an intestinal {alpha}E-DC (37) have been described. In lungs, CD4+ and CD8+ DC in the I-A+ and CD11c+ fraction were not detected (Fig. 2B, b and c). Lung CD11c+ 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, {alpha}E-DC, and PDC were found in lungs.


Figure 2
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  		FIGURE 2. Lung {alpha}E-DC is distinct from intraepithelial lymphocytes and other DC subsets. CD11c+ cells were isolated from lung single cell suspensions from naive mice by magnetic cell sorting. The retained CD11c+ cells and flow-through CD11c– cells were analyzed for DC subset marker expression. A–C, Cells were gated on 7-aminoactinomycin D-negative live cells and CD11c+ cells. D, Stained CD11c– flow-through cells were gated on the lymphoid population in forward scatter vs side scatter dot plots, 7-aminoactinomycin D-negative live cells, and CD3+ cells for T cell staining. A total of 10,000 cells were collected for each analysis. A, {alpha}E-DC is distinct from PDC. Lung integrin {alpha}Ebeta7+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 on live cells and mPDCA-1+ cells. B, {alpha}E-DC (circled population) do not express CD4 and CD8 splenic DC markers. C, {alpha}E-DC is positive for both integrin {alpha}E and beta7 staining. B and C, CD11c+ lung cells were stained and analyzed as in A. D, Lung intraepithelial lymphocytes are CD11c– and I-A– or I-Alow. CD11c– lung flow-through cells gated on CD3+ cells were stained and analyzed as indicated. This experiment was repeated twice with similar results.

 
{alpha}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 {alpha}E-DC.

{alpha}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 {alpha}Ebeta7 integrin. As stated earlier, {alpha}E-DC express the integrin {alpha}Ebeta7 (Fig. 2C) but not lymphocyte Peyer’s patch high endothelial venule adhesion molecule 1 ({alpha}4beta7), whereas intraepithelial lymphocytes express both. {alpha}E-DC do not express CD3, TCR-{alpha}beta, and TCR-{gamma}{delta} (Fig. 2Ba; TCR expression not shown). The intraepithelial lymphocyte numbers in lung cell suspensions were analyzed (Fig. 2D). Lung digests contained 6.5% lymphocytes, 25% of which were CD3+ T cells (6 x 105 CD3+ cells per lung). Approximately 16% of the CD3+ T cells expressed {alpha}Ebeta7 integrin (Fig. 2D, b and c). Intraepithelial lymphocytes expressed no CD11c (Fig. 2Dd) and low levels of I-A by only a small percentage of the population (Fig. 2De), and thus are readily distinguishable from {alpha}E-DC. They were present in lower numbers in lungs than {alpha}E-DC. In absolute numbers, only ~9.0 x 104 intraepithelial lymphocytes as compared with 19 x 104 {alpha}E-DC were found per mouse lung. Between the CD4+ and CD8+ lung intraepithelial lymphocyte subsets, more CD8+ intraepithelial lymphocytes were found and the ratio of CD8+/CD4+ intraepithelial lymphocyte was 1.4 (Fig. 2Dc). The absolute cell numbers were 5.5 x 104 and 3.8 x 104 per lung for CD8+ and CD4+ T cells, respectively. The CD8+ intraepithelial lymphocytes that make up 40% of all lung CD8+ T cells and 8.6% of CD3+ T cells likely represent the major CD8{alpha}{alpha} intraepithelial lymphocyte population (39). Although TCR{gamma}{delta}+ T cells were important in mucosal immunoregulation, few {gamma}{delta}+ T cells were found. They comprised only 2.8% of total CD3+ T cells, but 35% of {gamma}{delta}-T cells were {alpha}Ebeta7 integrin positive. The remaining lung T cells were TCR{alpha}beta+.

Adhesion molecules are highly expressed on {alpha}E-DC

Other adhesion molecules besides integrin {alpha}Ebeta7 are required for the migration of {alpha}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, CD11c+ DC were stained with either FITC-conjugated anti-CD103 or anti-I-A mAb and PE-conjugated anti-adhesion molecule mAb. {alpha}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). {alpha}E-DC were found to express the integrin subunits {alpha}4, {alpha}5, {alpha}v (CD51), and beta3 (CD61) (Fig. 3B, Table I). These molecules form heterodimers that function in DC migration into lungs or bind to extracellular matrix proteins such as laminin and collagen. {alpha}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).


Figure 3
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  		FIGURE 3. Surface marker expression on {alpha}E-DC. CD11c+ lung cells isolated by magnetic cell sorting were stained and analyzed as indicated in the dot plots. Cells were gated on 7-aminoactinomycin D-negative live cells and CD11c+ cells. The {alpha}E-DC populations were circled. Bd, the Mac-3+ macrophages were also circled. In I-A-stained cells (A, c and d; B, e–h; and C, f–h), the cell populations with the lower I-A staining is {alpha}E-DC, as shown in Ac. All analyses were from the same experiment, and four similar experiments have been performed. The mean fluorescence intensities for the Ag on the y-axis for {alpha}E-DC are shown in each panel.

 

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  		Table I. Activation of lung {alpha}E-DC in mice with asthma-like diseasea

 

Figure 5
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  		FIGURE 5. Pinocytosis of FITC-dextran by lung {alpha}E-DC, CD11bhigh-DC, and macrophages. CD11c+ 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. +m represents plus mannan. E, A separate experiment showing FITC-dextran uptake by {alpha}E-DC, CD11bhigh-DC, and macrophages at 60 min is shown. a, The white arrows show an {alpha}E-DC and the white triangle marks a putative CD11bhigh-DC. Bars show 5 µm. Similar pinocytosis experiments have been performed three times.

 
{alpha}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 {alpha}E-DC was examined by flow cytometry. Among a panel of costimulation molecules examined, {alpha}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 products, was also examined. Interestingly, {alpha}E-DC expressed TLR2, which responds to lipoteichoic acid and peptidoglycan but not TLR4, which responds to LPS (Fig. 3C, g and h).

Lung macrophages express high levels of B7-H1 and Siglac-F

Lung macrophages constitute a major CD11c+ population (Fig. 1B). Their distinguishing surface markers were sought to facilitate the comparison of their functions with DC in CD11c+ lung cell populations. In earlier experiments, lung macrophages were found 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 90% of the macrophages in the CD11c+ fraction (population 1 in Fig. 1Ab) were recovered by F4/80-magnetic microbead selection, and both the selected and residual macrophages in the flow-through were found to express the same surface markers. A small percentage (16%) of these macrophages expressed low levels of I-A (compare Fig. 4A, a with b). Lung macrophages expressed high levels of CD11c (Fig. 4Ac). Besides expressing F4/80, they expressed high levels of two new surface markers, the inhibitory costimulation molecule B7-H1 (42) and the sialic acid-binding lectin Siglac F, which is also highly expressed by eosinophils and immature bone marrow macrophages (Fig. 2, d–f) (43, 44). A subpopulation of these macrophages was also positive for Mac-3 (Fig. 3Bd, population M). Thus three specific markers, F4/80, B7-H1, and mSiglac-F, are useful for lung macrophage identification. Because B7-H1 is an inhibitory costimulation molecule (42), the high expression of B7-H1 on lung macrophages may explain the inhibitory function of pulmonary macrophages in T cell responses (45).


Figure 4
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  		FIGURE 4. Characterization of lung macrophages and PDC. A, Pulmonary macrophages were isolated by anti-F4/80-magnetic microbeads and gated on 7-aminoactinomycin D-negative live cells and CD11c+ cells. The cells were stained and analyzed as indicated. B, PDC were enriched by anti-mPDCA-1-magnetic microbeads, gated on 7-aminoactinomycin D-negative live cells and mPDCA-1+ cells (a), and analyzed for DC subset-specific markers or costimulation molecules. These isolated PDC were also positive for B220 and Gr-1 as shown in Fig. 2A, e and f. The mean fluorescence intensities for the Ag on the y-axis for encircled macrophage and PDC populations are shown in the dot plots. Two similar analyses with the same results have been performed for each cell type.

 
Lung PDC express low levels of costimulation molecules

PDC may play a significant role in immune responses in lungs. The presence of PDC has been shown in lungs, but the DC population has not been characterized (22, 28, 29). To examine lung PDC surface marker expression, these cells were first enriched with anti-mPDCA-1-magnetic microbeads followed by staining with anti-I-A and other surface markers (Fig. 4B). In three experiments, 6.2 ± 2.6 x 104 PDC were isolated per lung. Besides PDC, the anti-mPDCA-1 mAb also stained some other unspecified lung cell populations (Fig. 4Bc). However, PDC can be identified readily as a discrete population with low autofluorescence, intermediate I-A expression (Fig. 2, Ad and Fig. 4, Bb), and high levels of B220 and Gr-1 expression (Fig. 2A, e and f). PDC expressed no {alpha}E (Fig. 4Bd), but low to intermediate levels of CD11b (Fig. 4Be). No detectable amounts of B7-1 and CD40, and low levels of B7-2 and B7-DC were found on their surface (Fig. 4B, f–i). Thus substantial numbers of PDC were found in mouse lungs, but they express low numbers of costimulation molecules.

{alpha}E-DC pinocytose avidly

An essential function of DC is Ag uptake. The pinocytosis of FITC-dextran by lung DC subsets was examined (Fig. 5). FITC-dextran uptake by {alpha}E-DC was readily observed (Fig. 5Aa). These DC pinocytosed avidly with an initial burst of uptake in the first 20 min (Fig. 5, Ab and D). This higher initial uptake rate was not seen in CD11bhigh-DC, which pinocytosed at a linear but faster rate for ~40 min before starting to reach plateau (Fig. 5D). The rapid FITC-dextran uptake by {alpha}E-DC was not due to mannose receptor-mediated uptake because a 10-fold excess of S. cerevisiae mannan failed to reduce FITC-dextran uptake rate appreciably (Fig. 5, Ac and D). The lack of mannose receptor involvement was also true for FITC-dextran uptake by CD11bhigh-DC and macrophages (Fig. 5, Bc, Cc, and D). The finding was further supported by the failure of anti-mannose receptor (CD204) mAb to stain {alpha}E-DC (data not shown). Although {alpha}E-DC pinocytosed less rapidly compared with CD11bhigh-DC, their pinocytic rate was comparable to that of macrophages, which are known to pinocytose rapidly (compare Fig. 5, A with B and C). The uptake of FITC-dextran was also readily observed in lung DC and macrophages by confocal microscopy (Fig. 5E).

{alpha}E-DC stimulate T cell proliferation efficiently

The potency of {alpha}E-DC in stimulating T cell proliferation was examined by using purified lung DC populations sorted by flow cytometry. The sorted populations were 98% pure and showed no overlap in marker staining between the two DC populations (Fig. 6A). Both {alpha}E-DC and CD11bhigh-DC stimulated DO11.10 T cell proliferation efficiently when either soluble anti-CD3 mAb or OVA323–338 peptide was used as a stimulant (Fig. 6B). The stimulation potencies of {alpha}E-DC and CD11bhigh-DC were comparable, and they were at least 20-fold more potent than irradiated splenocytes on a per cell basis.


Figure 6
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  		FIGURE 6. Ag presentation and IL-12 production by FACS-sorted pure {alpha}E-DC and CD11bhigh-DC populations. Sorted {alpha}E-DC or CD11bhigh-DC populations (A, 98% pure) were used to stimulated purified CD4+ splenic T cells from DO11.10 transgenic mice with either anti-CD3 (Ba) or OVA323–338 peptide (Bb) according to Materials and Methods and compared with stimulation by irradiated splenocytes. The production of IL-12 by the DC subsets after 36 h of stimulation with the indicated TLR ligands or anti-CD40 mAb is shown in C. A representative experiment from three similar experiments is shown.

 
{alpha}E-DC produce IL-12

Lymph node DC subsets have been shown to have different cytokine production profiles (46). Lung {alpha}E-DC and CD11bhigh-DC production of the signature cytokines IL-12, IFN-{alpha}, and IFN-{gamma} for different DC subsets were measured. Because flow cytometry analysis showed that lung DC expressed TLR2 (Fig. 3Cg) and that efficient DC stimulation required the activation by multiple TLR ligands (47), highly purified DC populations (Fig. 6A) were incubated with combinations of ligands of different TLR or anti-CD40 mAb with the TLR2 ligand PAM3CSK4. No IFN-{alpha} or IFN-{gamma} were detected in DC culture supernatants. However, IL-12 production was readily detected. Control cultures with no stimulants produced low levels of IL-12 (Fig. 6C). There were significant differences in responses between {alpha}E-DC and CD11bhigh-DC to the stimulants. PAM3CSK4 combined with poly(I:C) were the strongest stimulants for {alpha}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 {alpha}E-DC had a higher response to TLR3 ligands than CD11bhigh-DC and both DC types are similar to the CD8+CD4– splenic DC subset in their ability to produce IL-12 (46).

{alpha}E-DC are localized in the airway mucosa and perivascular region

{alpha}E-DC are distinguishable from intraepithelial lymphocytes by their CD11c and I-A expression (Fig. 2D, d and e). Although eosinophils are beta7 integrin+, they are CD11c-low and I-A– (data not shown). Thus colocalization of the combinations of {alpha}E or beta7 integrin with either CD11c or I-A staining constitutes a reliable criterion for identifying {alpha}E-DC. Confocal microscopy of lung tissues stained with combinations of mAb against {alpha}E, beta7, CD11c, or I-A showed that these markers were colocalized and that {alpha}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, {alpha}E-DC were found tightly apposed to the basal surface of bronchial epithelial cells where the {alpha}Ebeta7 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 {alpha}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). {alpha}E-DC constituted 70–75% of the I-A+CD11c+-DC in the proximal subepithelial and periarterial regions. Unlike PDC, {alpha}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 CD11b+ cells representing neutrophils or eosinophils were also present in the mucosal as well as the alveolar regions (Fig. 7Cc, arrow).


Figure 7
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  		FIGURE 7. Confocal microscopy of {alpha}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 Cf is shown in the inset. Arrows show CD11bhigh-DC (yellow to white image). D and E, Anti-I-A and anti-beta7 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 Langerin+ (D) or Claudin-1+ (E) cells. Photomicrographs were captured with a x40 objective and bars show 20 or 50 µm as indicated. ar, Arteriole; br, bronchiole; and epi, bronchiolar epithelium. These experiments have been repeated four times with different tissues from different experiments.

 
{alpha}E-DC expresses the epidermal DC marker Langerin

To support the epithelial nature of {alpha}E-DC, an Affymetrix microarray database obtained from mRNA of sorted {alpha}E-DC and CD11bhigh-DC was constructed and used to search for differentially expressed genes. The mRNA of Langerin, an epidermal Langerhans cell marker shown to participate in Birbeck granule formation and nonpeptide Ag presentation (48, 49), was highly expressed in {alpha}E-DC, and its expression is 12-fold higher in {alpha}E-DC than in CD11bhigh-DC (Table II). The microarray data were confirmed by real-time PCR, which also showed that {alpha}E-DC expressed much higher (23-fold) Langerin mRNA than CD11bhigh-DC (Table II). The Langerin mRNA product with 40 cycles of amplifications migrated with the expected mobility (Fig. 8, lanes 3 and 4). Staining of lung tissues by anti-Langerin Ab showed that Langerin staining was only colocalized with {alpha}E-DC, which showed bright I-A and integrin beta7 staining and are localized mostly either in the epithelium or arteriolar wall (Fig. 7D). The results confirmed that {alpha}E-DC expressed Langerin and that it was the predominant cell type in lungs for this expression. The Langerin expression of {alpha}E-DC suggests that they are similar to Langerhans cells, CD8+ lymphoid DC, and DEC-205highCD8low lymph node DC, all of which express this protein (50, 51).


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

 

Figure 8
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  		FIGURE 8. Amplified Langerin and tight junction protein cDNA products migrated with the expected mobilities. cDNA transcribed from total RNA of sorted {alpha}E-DC (lanes a) or CD11bhigh-DC (lanes b) were amplified for 40 cycles as described in Materials and Methods. PCR products were fractionated in 1.8% agarose gels. The expected product sizes were: CD207 (Langerin), 178 bp; Claudin-1, 151 bp; Claudin-7, 213 bp; ZO-2, 171 bp; and beta-actin, 181 bp. Lanes 1 and 2 contained no cDNA. This analysis was performed twice.

 
{alpha}E-DC express Claudin-1, Caludin-7, and ZO-2

To gain access to Ag or pathogens in the airways, {alpha}E-DC in the mucosa must traverse the bronchial epithelial layer through the tight junction barrier. This is achieved by the {alpha}E-DC surface expression of tight junction proteins, which interact with those on the epithelial cells and allow the cell body to squeeze through (reviewed in Ref.52). Differential discovery of Affymetrix microarray data showed that among the known tight junction proteins, Claudin-1, Claudin-7, and ZO-2 mRNA were overexpressed in {alpha}E-DC compared with CD11bhigh-DC (Table II). Further examination of mRNA levels of the major tight junction proteins zona occludins (tight junction proteins), Claudins, and junction adhesion molecules showed that except ZO-2, Claudin-1, and Claudin-7, {alpha}E-DC and CD11bhigh-DC expressed none or very low levels of the mRNA for these groups of proteins in the microarray data set (data not shown). Real-time PCR confirmed that {alpha}E-DC expressed much higher levels of Claudin-1 and Claudin-7 and somewhat higher level of ZO-2 than CD11bhigh-DC (Table II). The PCR products exhibited the expected migration mobilities (Fig. 8, lanes 5–10). Staining of lung tissues for Claudin-1 and Claudin-7 showed that these tight junction proteins are expressed by {alpha}E-DC (Claudin-1, Fig. 7E; Claudin-7 is specifically expressed in {alpha}E-DC but is dim and, therefore, not shown) and the staining is colocalized with I-A and beta7 in the epithelia or vascular walls. These results showed that {alpha}E-DC are equipped with tight junction proteins that will enable them to migrate or extend their pseudopods into the bronchiolar luminal space to capture Ag for processing and presentation.

Lung {alpha}E-DC accumulation and activation in mice with Ag-induced inflammation

The role of lung {alpha}E-DC in asthma was examined in an OVA-induced asthma-like model (33). Immunized mice exhibited marked airway hyperresponsiveness and eosinophilia. {alpha}E-DC increased markedly, from an average of 1.9 x 105 in control mice to 5.4 x 105 {alpha}E-DC per lung in immunized mice. They constituted 21% of the total lung I-AhighCD11chigh-DC. The increase was not 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 was found by day 7, when the label was found exclusively associated with macrophages in the paracortical region of thoracic lymph node, likely representing residual fluorescent material from phagocytosed exogenous DC. The second is that when 1 x 106 CD45.2 bone-marrow-derived DC were injected intratracheally into CD45.1 mice, exogenous DC constituted <1% of the total CD11c+ lung cells in unchallenged mice on day 6. These injected cells expressed no {alpha}E. In OVA-immunized mice, {alpha}E-negative lung DC increased by at least 12-fold compared with controls. Thus exogenous DC constituted at the most 0.7% of the {alpha}E-negative DC. This number is most likely to be much lower because of cellular activation, mobilization, and apoptosis in the inflamed lung. The third line of evidence is that, in the study by van Rijt et al. (6) using a similar immunization protocol, no residual exogenous DC was detected. {alpha}E-DC in asthma-induced mice were activated, with significant increases in surface expression of B7-2, B7-DC, CD40, and CD49d expression (Table I). ICAM-1 and MHC class II expression also showed consistent increases in intensities on {alpha}E-DC. However, the increase did not reach statistical significance because of the high scattering of the results from replicate experiments.

Large numbers of {alpha}E-DC and CD11bhigh-DC were present in the mucosa of inflamed lungs

Confocal microscopy showed that as in control lungs, large numbers of {alpha}E-DC and CD11bhigh-DC were present in the airway mucosa, adjacent to the vascular walls, and within the leukocyte infiltration areas (Fig. 7B and 7C, d–f). Many more CD11bhigh-DC were found (Fig. 7C), in agreement with the increases in lung CD11bhigh-DC numbers (12-fold higher) in immunized mice. Interestingly, the preferential localization of {alpha}E-DC and CD11bhigh-DC were different. {alpha}E-DC remained largely immediately adjacent to the basal lamina of the bronchial epithelia and arterioles, where 65–70% of the I-A+CD11c+-DC were integrin {alpha}Ebeta7+ (Fig. 7B). CD11bhigh-DC, in contrast, were found in the proximal subepithelia and vascular wall as a minor I-A+CD11c+ population (Fig. 7C, e and f). They occurred primarily interspersed within the leukocyte infiltration zone of the peribronchial and perivascular cuffs (Fig. 7Cf, inset, arrows). The results suggest that {alpha}E-DC and CD11bhigh-DC exhibit different preferential localization.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Although DC are considered the critical cell type for airway immunity (3, 4, 5, 6), lung DC subsets are poorly characterized. With the combination of magnetic microbead sorting followed by FACS purification, we obtained 5 x 103 each of 98% pure {alpha}E-DC and CD11bhigh-DC (1 x 104 DC per mouse total), a 4-fold increase in yield regarding I-Ahigh lung DC when compared with previous reports (23, 27, 28). Besides myeloid DC, PDC have been described in lung cell digests, but the numbers are too small for phenotypic and functional analyses (22, 28). Furthermore, von Garnier et al. have detected PDC in their CD11clow and CD11c– lung cell fractions that are not normally analyzed for DC occurrence, and PDC numbers are small in the CD11c+ lung cell population (29). The yield of PDC by anti-CD11c-magnetic microbead isolation in our hands was similarly variable. Using magnetic beads specific for PDC, we were able to enrich for PDC in lung digests. The yield was estimated to be 6 x 104 PDC per lung. Thus PDC occurrence is approximately one-third that of either {alpha}E-DC or CD11bhigh-DC in lungs. CD8+ and CD4+ lymphoid DC have also not been identified in lungs. Our results showed that few if any of these lymphoid DC were present in control (Fig. 2B) and inflamed lungs (data not shown). It is noteworthy that the migrating airway DC that have captured intratracheally introduced FITC-OVA were reported to be CD8+ (13). The results suggest that the CD8 marker is induced in lung DC upon activation and migration to the lymph node. Epidermal Langerhans cells have been shown to express CD8 similarly at a low to intermediate level (16). Thus in lungs, three major I-A+ DC types have been identified in this study. In addition to these three subsets, a mucosal CD11bhigh-DC population in the respiratory tract with high turnover rate has also been identified (29).

{alpha}E-DC have not been described in lungs previously; however, they have been found in the mesenteric and other lymph nodes (37) and in rat small intestine lamina propria (32, 38). In the skin-draining lymph node, integrin {alpha}Ebeta7-expressing DC have also been isolated (17). However, these DC are postulated to have acquired their {alpha}Ebeta7 integrin in the draining lymph node and thus may be different from those found in the lung mucosa. Lung {alpha}E-DC are clearly distinct from intraepithelial lymphocytes by their lack of CD3, TCR{alpha}beta, TCR{gamma}{delta}, CD4, or CD8 expression (Fig. 2B), and by the absence of CD11c and I-A on intraepithelial lymphocytes (Fig. 2D, d and e). They are two to three times more numerous than intraepithelial lymphocytes in the lung and may be in even higher percentages in inflamed lungs. In the lung epithelia and arteriolar walls, most integrin {alpha}E+ or beta7+ cells were I-A+ DC (Fig. 7). The majority of the intraepithelial lymphocytes in lungs (60%) were the CD8+ T cells, which likely represent the CD8{alpha}{alpha}+TCR{alpha}beta+ intraepithelial lymphocytes. These cells were regulatory in function and may have relevance in asthma pathogenesis (53).

In this report, we have identified the mucosal {alpha}E-DC as a major DC population in mouse lungs. They are distinct from the previously described major lung myeloid CD11bhigh-DC population (28) with regard to surface marker expression. Compared with {alpha}E-DC, CD11bhigh-DC express no {alpha}Ebeta7, higher levels of I-A, much higher levels of CD11b, and lower level of CD11c. In inflamed lungs, stimulated CD11bhigh-DC expressed much higher levels of B7-H1 and B7-DC than {alpha}E-DC (data not shown). There were also functional differences between these two DC subsets. {alpha}E-DC pinocytosed at a lower rate, but with an initial burst that is absent with CD11bhigh-DC (Fig. 5). Furthermore, {alpha}E-DC responded to TLR3 stimulation with a higher production of IL-12 (Fig. 6C). In terms of tissue occurrence, {alpha}E-DC are preferentially localized at the basal lamina of the bronchial epithelia and arterioles (Fig. 7, A and B), whereas most of the CD11bhigh-DC are present more distal to the basal lamina, and are within the leukocyte infiltrating area in inflamed lungs (Fig. 7C). {alpha}E-DC also express Langerin and markedly higher levels of tight junction proteins. These results support the hypothesis that {alpha}E-DC and CD11bhigh-DC are distinct DC populations.

Several lines of evidence suggest that {alpha}E-DC exhibit phenotypic and functional characteristics similar to that of lymphoid DC. The first is that {alpha}E-DC express Langerin. Langerin mRNA has been detected in the CD8highCD11blow population of spleen and LN cells (51). Secondly, among CD11chigh splenic DC, integrin {alpha}E mRNA was expressed only in CD8{alpha}+, but not in CD4+ or double-negative DC subsets (54). The third is that in microarray analyses, several genes have been found to be expressed at much higher levels in {alpha}E-DC than in CD11bhigh DC. These genes include CD24a (14-fold), Notch 4 (11-fold), and IFN consensus sequence binding protein 1 (8-fold) (S. J. Sung, unpublished results). Similar overexpression of these genes by CD8+ DC in gene chip data has been reported when splenic CD8+ DC mRNA expression was compared with that of either CD4+ or double-negative splenic myeloid DC (54). The differential overexpression of Notch 4 mRNA by {alpha}E-DC is of considerable interest because of the regulation of Th1 and Th2 responses by Notch signaling in T cells (55) and the relevance of Th2 cells in asthma. Of the four Notch genes, detectable mRNA expression in freshly isolated lung CD11c+ DC was observed only for Notch 1 and Notch 4 by microarray analysis. The signal intensities for Notch 1 mRNA were 106 ± 7 and 197 ± 59 for {alpha}E-DC and CD11bhigh-DC, respectively, and the corresponding values for Notch 4 mRNA were 629 ± 93 and 39 ± 3 (S. J. Sung, unpublished results). Notch signaling has been shown to induce the differentiation of hemopoietic precursors into myeloid DC precursors (56) and the maturation of human monocyte-derived DC (57). However, the significance of the Notch 4 expression by {alpha}E-DC in modulating lung immune response has yet to be determined. The expression of mRNA for the Th subset-directing Notch receptor ligands Jagged 1, Jagged 2, and Delta 1 by these lung DC in microarrays were also examined. Although little Delta 1 mRNA was observed, detectable levels of Jagged 1 (75 ± 25, 148 ± 57) and Jagged 2 (82 ± 20, 41 ± 9) mRNA were observed in {alpha}E-DC and CD11bhigh-DC, respectively. It is likely that the mRNA and protein expression of these Notch receptor ligands are induced upon lung DC activation. It will be important to determine whether DC Notch ligand expression and stimulation is a key mechanism for directing Th2 responses in asthma.

The difference in cellular properties between {alpha}E-DC and CD11bhigh-DC suggests that these two DC subsets may serve different functions in lung inflammation. {alpha}E-DC are tightly apposed to the basolateral side of bronchial epithelial cells where E-cadherin, the ligand for {alpha}Ebeta7, occurs (Fig. 7). Coupled with their surface expression of tight junction proteins such as Claudin-1, Caludin-7, and ZO-2, {alpha}E-DC can open up tight junctions between epithelial cells and either send their dendrites or directly migrate across the bronchial epithelium into the airway for Ag capture, as have been demonstrated for intestinal DC (58). Because {alpha}E-DC constitutes 75% of the DC population immediately adjacent to the epithelia, they are likely to be the DC population observed to form an I-A+ reticular network in the airway mucosa (10). In inflamed lungs, {alpha}E-DC remained the main DC population in the epithelium, despite a much larger increase in lung CD11bhigh-DC. These observations suggest that a major function of {alpha}E-DC may be to capture Ag in the airways and transport the Ag to the thoracic lymph node for T cell priming. CD11bhigh-DC, in contrast, are found interacting with other leukocyte cell types in the peribronchial and perivascular leukocyte infiltrating areas. They may be responsible for activating infiltrating leukocytes. CD11bhigh-DC may also be important for leukocyte recruitment. DC have been shown to produce large amounts of CC and CXC chemokines such as thymus- and activation-regulated chemokine, macrophage-derived chemokine, and IFN-inducible protein 10 (59). The interaction of CD11bhigh-DC with other inflammatory leukocytes may induce high levels of chemokines for further leukocyte recruitment. Thus there may a dichotomy of functions for {alpha}E-DC and CD11bhigh-DC, the former being responsible for airway Ag transport to the draining lymph node and the latter for propagating the local lung inflammatory response. If the hypothesis is correct, then {alpha}E-DC will be more mobile while CD11bhigh-DC will preferentially remain in lungs during Ag challenges.

Although {alpha}E-DC may play a key role in capturing Ag in the airway, they may also be important in capturing pathogenic Ag in the interstitium and in interacting with infiltrating leukocytes in the perivascular and peribronchial cuffs. The finding that primed T cells interact with DC in the epithelium suggests that T cell-DC interaction in the lungs is a significant and common event (14, 15). However, the full extent of the consequences of these interactions in lungs is unknown. Because of the rapid turnover of lung DC (10), there will be large numbers of DC in transit between the vasculature and the mucosa and between the mucosa and the lymphatic ducts at any instant for Ag or cellular encounters. Furthermore, the trophic properties of Ag and pathogens leading to the release of chemotactic factors such as f-Met-Leu-Phe, complement fragments such as C5a, and TLR ligands will induce the migration of DC toward the attractant source. In addition to the mucosal {alpha}E-DC, many {alpha}E-DC are also present on the parenchymal side of the pulmonary arterioles with ready access to the interstitium (Fig. 7, A and B). The exact significance of this vascular localization for {alpha}E-DC is unclear. However, an earlier report has also shown that DC are enriched on the luminal side of the vascular wall (60). These blood vessel-associated DC may serve in recruitment of leukocytes into the lung perivascular region by the production of chemokines.

Besides bearing DC hallmark surface Ag such as CD11c and MHC class II molecules, {alpha}E-DC are proficient in performing the three key DC functions, Ag uptake by fluid-phase pinocytosis, Ag presentation to T cells, and cytokine production (Figs. 5 and 6). These three DC functions have not been demonstrated for {alpha}E-DC in previous studies (17, 32, 37, 38). Two points regarding pinocytosis are noteworthy. First, CD11bhigh-DC pinocytosed at a faster rate than either {alpha}E-DC or macrophages. Secondly, there seemed to be a burst of pinocytosis in the initial 20 min of incubation by {alpha}E-DC but not CD11bhigh-DC. The initial pinocytic burst may indicate that {alpha}E-DC uses macropinocytosis to engulf large volumes of fluid rapidly (61). The finding that mannan did not substantially reduce FITC-dextran uptake support the notion that under the pinocytic condition of high FITC-dextran concentration, fluid-phase rather than receptor-mediated pinocytosis was measured. However, it is interesting that mannose receptor was not detected on these cells. This expression may be inducible by stimulants such as TLR ligands or cytokines in an inflammatory situation. With regard to interacting with carbohydrate ligands and with pathogens, these DC subsets are found to express mRNA for many C-type lectin receptors including DC-SIGN (CD209a) and, therefore, are capable of such recognition (S. J. Sung, unpublished results).

Compared with lung CD11bhigh-DC, {alpha}E-DC expressed most adhesion molecules at substantially lower levels. The notable exceptions are ICAM-1 and integrin {alpha}Ebeta7, which are expressed at high levels on {alpha}E-DC and thus may function as the key adhesion molecules determining {alpha}E-DC migration. {alpha}E-DC stimulate T cells efficiently by their expression of costimulation and accessory molecules such as B7-1 and B7-2. In immunized mice, {alpha}E-DC expressed significantly higher levels of the costimulation molecules B7-2, B7-DC, and CD40 (Table I). A large panel of other mAb against Ag including ICOSL, CD27L, CD30L, CD40L, 4-1BBL, and Ox40L has failed to stain {alpha}E-DC, indicating that B7-1, B7-2, B7-H1, B7-DC, and CD40 may be the key accessory molecules for their efficient Ag presentation function. The high expression of B7-DC by DC is relevant in allergic diseases. A stimulating anti-B7-DC mAb has been shown to inhibit allergic diseases (62). Although the mechanism for this blockage is presently unclear, the study indicates that B7-DC on DC plays a major role in lung antigenic responses.

This report is the first to provide clear evidence that {alpha}E-DC is a major DC type in lungs. {alpha}E-DC are clearly distinct from intraepithelial lymphocytes and other lung DC subsets, occur in large numbers in the airway mucosa, function efficiently in Ag uptake and presentation, produce IL-12 upon TLR stimulation, express tight junction proteins that allow them to traverse the bronchial epithelium readily, and increase markedly in number during lung antigenic challenges. Studying the migration of this DC type during antigenic challenge and the functional role of this cell type in lung inflammation will likely provide mechanistic insight on the pathogenesis of lung diseases.


   Acknowledgments
 
We thank Wei Shan, Elise Hackett, Binru Wang, Runpei Wu, Waunema Smith, and Natalie Walker for technical assistance, Hao M. Qian for advice, and A. Melissa Loggans for manuscript preparation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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 with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by National Institutes of Health Grants HL070065, HL65344, AR45222, and AI36938. Back

2 Address correspondence and reprint requests to Dr. Sun-Sang J. Sung, Division of Rheumatology and Immunology, Department of Internal Medicine, Box 800412, University of Virginia Health Sciences Center, Charlottesville, VA 22908. E-mail: sjs5c{at}Virginia.EDU Back

3 Abbreviations used in this paper: DC, dendritic cell; {alpha}E-DC, integrin {alpha}Ebeta7+ DC; PDC, plasmacytoid DC. Back

Received for publication September 21, 2005. Accepted for publication December 2, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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