A Major Lung CD103 (α_E)-β₇ Integrin-Positive Epithelial Dendritic Cell Population Expressing Langerin and Tight Junction Proteins

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 β₇, 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-γ, IL-12 p70, and IFN-α 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 αE-DC and CD11b^high-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 × 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-FcγRII/FcγRIII 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-FcγRII/FcγRIII 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 × 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-A^highCD103⁺, I-A^highCD11b^high, and I-A⁺Siglac-F⁺ cells representing αE-DC, CD11b^high-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 αE-DC or CD11b^high-DC and either 2 μg/ml anti-CD3 mAb or 5 μM OVA_323–339 peptide. Total splenocytes irradiated for 25 Gy were used as control APC.

Microarray analysis of αE-DC and CD11b^high-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-A^highCD103⁺CD11c⁺CD11b^low (αE-DC) and the I-A^highCD103⁻CD11c⁺CD11b^high (CD11b^high-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 αE-DC and CD11b^high-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 β-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).

αE-DC and CD11b^high-DC are the two major populations of I-A^high lung DC

Previous studies have identified the CD11b^high-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-A^high DC populations based on I-A and CD11c expression levels were found (Fig. 1⇓Ab, 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.

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FIGURE 1.

Identification of I-A^highCD11c^high 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-A^highCD11c⁺ 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 αE-DC and CD11b^high-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-A^high DC populations further. The CD11b^high population characterized in earlier studies (28) was found to constitute the major I-A^high DC population that expresses slightly higher I-A and lower CD11c (population 3 in Fig. 1⇑Ab, 11b population in B, b–d). Because lung contains an extensive epithelial surface, it is likely that the αE-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-A^high DC (Fig. 1⇑Ab). This hypothesis was found to be indeed the case. The lung I-A^high population with slightly higher CD11c but lower I-A expression (population 2 in Fig. 1⇑Ab) was found to be integrin αE⁺ (αE population in Fig. 1⇑B, b and c) but did not express lymphocyte Peyer’s patch high endothelial venule adhesion molecule 1 (α₄β₇; data not shown). This population has a low to intermediate surface CD11b expression (Fig. 1⇑Bb). However, a small population of these αE-DC (≈5% of αE⁺ cells) was CD11b^high representing perhaps activated αE-DC.

Large numbers of the two I-A^high DC types could be isolated by magnetic bead sorting. αE-DC and CD11b^high-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 αE-DC and CD11b^high-DC were 1.9 ± 1.2 × 10⁵ and 1.7 ± 0.5 × 10⁵ cells per mouse lung, respectively. Macrophages were recovered at 5.4 ± 3.9 × 10⁵ cells per lung. In normal lungs, the three major CD11c⁺ populations αE-DC, CD11b^high-DC, and macrophages comprised 14 ± 6.7%, 12.5 ± 1.4%, and 54 ± 3.9%, respectively, of the total CD11c⁺ cells. Thus αE-DC is a major lung DC population, comprising 40–60% of the lung CD11c⁺I-A^high DC.

αE-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 αE-DC (37) have been described. In lungs, CD4⁺ and CD8⁺ DC in the I-A⁺ and CD11c⁺ fraction were not detected (Fig. 2⇓B, 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 CD11b^high-DC, αE-DC, and PDC were found in lungs.

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FIGURE 2.

Lung αE-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, αE-DC is distinct from PDC. Lung integrin α_Eβ₇⁺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, αE-DC (circled population) do not express CD4 and CD8 splenic DC markers. C, αE-DC is positive for both integrin α_E and β₇ 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-A^low. CD11c⁻ lung flow-through cells gated on CD3⁺ cells were stained and analyzed as indicated. This experiment was repeated twice with similar results.

αE-DC is clearly distinct phenotypically from PDC. They express few of the PDC surface markers B220 and Gr-1 (Fig. 2⇑A, b and c). In contrast, PDC express high levels of B220 and Gr-1 on their surface (Fig. 2⇑A, e and f). In data not shown, mPDCA-1 was also absent on αE-DC.

αE-DC do not express the lymphoid markers CD4 and CD8 expressed by some DC subsets in lymphoid tissues (Fig. 2⇑B, b and c). It is also distinct from the mucosal intraepithelial lymphocytes, which are found in lungs and also express the α_Eβ₇ integrin. As stated earlier, αE-DC express the integrin α_Eβ₇ (Fig. 2⇑C) but not lymphocyte Peyer’s patch high endothelial venule adhesion molecule 1 (α₄β₇), whereas intraepithelial lymphocytes express both. αE-DC do not express CD3, TCR-αβ, and TCR-γδ (Fig. 2⇑Ba; TCR expression not shown). The intraepithelial lymphocyte numbers in lung cell suspensions were analyzed (Fig. 2⇑D). Lung digests contained 6.5% lymphocytes, 25% of which were CD3⁺ T cells (6 × 10⁵ CD3⁺ cells per lung). Approximately 16% of the CD3⁺ T cells expressed α_Eβ₇ integrin (Fig. 2⇑D, b and c). Intraepithelial lymphocytes expressed no CD11c (Fig. 2⇑Dd) and low levels of I-A by only a small percentage of the population (Fig. 2⇑De), and thus are readily distinguishable from αE-DC. They were present in lower numbers in lungs than αE-DC. In absolute numbers, only ∼9.0 × 10⁴ intraepithelial lymphocytes as compared with 19 × 10⁴ αE-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. 2⇑Dc). The absolute cell numbers were 5.5 × 10⁴ and 3.8 × 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 CD8αα intraepithelial lymphocyte population (39). Although TCRγδ⁺ T cells were important in mucosal immunoregulation, few γδ⁺ T cells were found. They comprised only 2.8% of total CD3⁺ T cells, but 35% of γδ-T cells were α_Eβ₇ integrin positive. The remaining lung T cells were TCRαβ⁺.

Adhesion molecules are highly expressed on αE-DC

Other adhesion molecules besides integrin α_Eβ₇ are required for the migration of αE-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. αE-DC can be distinguished from CD11b^high-DC by CD103 positivity (Fig. 3⇓Ab) or lower I-A staining in flow cytometry dot plots (Fig. 3⇓, Ac and Be). αE-DC were found to express the integrin subunits α₄, α₅, α_v (CD51), and β₃ (CD61) (Fig. 3⇓B, Table I⇓). These molecules form heterodimers that function in DC migration into lungs or bind to extracellular matrix proteins such as laminin and collagen. αE-DC also express high levels of ICAM-1 (see Fig. 5⇓Bf) and some Mac-3 (Fig. 3⇓Bd). The latter binds to the macrophage galactose glycoconjugate-binding protein galectin-3 (Mac-2) (41).

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FIGURE 3.

Surface marker expression on αE-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 αE-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 αE-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 αE-DC are shown in each panel.

αE-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 αE-DC was examined by flow cytometry. Among a panel of costimulation molecules examined, αE-DC was found to express moderate levels of several B7 molecules including B7-1, B7-2, B7-H1, and B7-DC (Fig. 3⇑C, a–d; Table I⇑). They also expressed the accessory molecules CD40 and Ox40L, which are important for DC activation and costimulation (Fig. 3⇑C, e and f). The expression of TLR2 and TLR4, which are responsible for DC responses to bacterial products, was also examined. Interestingly, αE-DC expressed TLR2, which responds to lipoteichoic acid and peptidoglycan but not TLR4, which responds to LPS (Fig. 3⇑C, g and h).

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

Lung macrophages constitute a major CD11c⁺ population (Fig. 1⇑B). 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. 4⇓A). Greater than 90% of the macrophages in the CD11c⁺ fraction (population 1 in Fig. 1⇑Ab) 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. 4⇓A, a with b). Lung macrophages expressed high levels of CD11c (Fig. 4⇓Ac). 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. 3⇑Bd, 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).

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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. 2⇑A, 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. 4⇑B). In three experiments, 6.2 ± 2.6 × 10⁴ PDC were isolated per lung. Besides PDC, the anti-mPDCA-1 mAb also stained some other unspecified lung cell populations (Fig. 4⇑Bc). 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. 2⇑A, e and f). PDC expressed no αE (Fig. 4⇑Bd), but low to intermediate levels of CD11b (Fig. 4⇑Be). No detectable amounts of B7-1 and CD40, and low levels of B7-2 and B7-DC were found on their surface (Fig. 4⇑B, f–i). Thus substantial numbers of PDC were found in mouse lungs, but they express low numbers of costimulation molecules.

αE-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 αE-DC was readily observed (Fig. 5⇓Aa). 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 CD11b^high-DC, which pinocytosed at a linear but faster rate for ∼40 min before starting to reach plateau (Fig. 5⇓D). The rapid FITC-dextran uptake by αE-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 CD11b^high-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 αE-DC (data not shown). Although αE-DC pinocytosed less rapidly compared with CD11b^high-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. 5⇓E).

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FIGURE 5.

Pinocytosis of FITC-dextran by lung αE-DC, CD11b^high-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 αE-DC, CD11b^high-DC, and macrophages at 60 min is shown. a, The white arrows show an αE-DC and the white triangle marks a putative CD11b^high-DC. Bars show 5 μm. Similar pinocytosis experiments have been performed three times.

αE-DC stimulate T cell proliferation efficiently

The potency of αE-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. 6⇓A). Both αE-DC and CD11b^high-DC stimulated DO11.10 T cell proliferation efficiently when either soluble anti-CD3 mAb or OVA_323–338 peptide was used as a stimulant (Fig. 6⇓B). The stimulation potencies of αE-DC and CD11b^high-DC were comparable, and they were at least 20-fold more potent than irradiated splenocytes on a per cell basis.

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FIGURE 6.

Ag presentation and IL-12 production by FACS-sorted pure αE-DC and CD11b^high-DC populations. Sorted αE-DC or CD11b^high-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 OVA_323–338 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.

αE-DC produce IL-12

Lymph node DC subsets have been shown to have different cytokine production profiles (46). Lung αE-DC and CD11b^high-DC production of the signature cytokines IL-12, IFN-α, and IFN-γ for different DC subsets were measured. Because flow cytometry analysis showed that lung DC expressed TLR2 (Fig. 3⇑Cg) and that efficient DC stimulation required the activation by multiple TLR ligands (47), highly purified DC populations (Fig. 6⇑A) were incubated with combinations of ligands of different TLR or anti-CD40 mAb with the TLR2 ligand PAM₃CSK_4. No IFN-α or IFN-γ 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. 6⇑C). There were significant differences in responses between αE-DC and CD11b^high-DC to the stimulants. PAM₃CSK₄ combined with poly(I:C) were the strongest stimulants for αE-DC but had only moderate effects on CD11b^high-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 αE-DC had a higher response to TLR3 ligands than CD11b^high-DC and both DC types are similar to the CD8⁺CD4⁻ splenic DC subset in their ability to produce IL-12 (46).

αE-DC are localized in the airway mucosa and perivascular region

αE-DC are distinguishable from intraepithelial lymphocytes by their CD11c and I-A expression (Fig. 2⇑D, d and e). Although eosinophils are β₇ integrin⁺, they are CD11c-low and I-A⁻ (data not shown). Thus colocalization of the combinations of α_E or β₇ integrin with either CD11c or I-A staining constitutes a reliable criterion for identifying αE-DC. Confocal microscopy of lung tissues stained with combinations of mAb against α_E, β₇, CD11c, or I-A showed that these markers were colocalized and that αE-DC were present in large numbers mainly in the airway mucosa or on the parenchyma side of the arteriole walls (Fig. 7⇓A). In the mucosa, αE-DC were found tightly apposed to the basal surface of bronchial epithelial cells where the α_Eβ₇ integrin ligand E-cadherin is present. DC were rarely seen within the bronchiolar epithelial layer (Fig. 7⇓A). The intraepithelial network seen in airway tangential sections (10) likely represents the pseudopod extensions rather than the cell bodies of DC. The periarteriole αE-DC were found directly underneath the vascular endothelial cells, perhaps attached to the basal lamina (Fig. 7⇓, A, d–f, and B, d–f). αE-DC constituted 70–75% of the I-A⁺CD11c⁺-DC in the proximal subepithelial and periarterial regions. Unlike PDC, αE-DC were rarely observed in the alveolar septa. Some CD11b^high-DC characterized by bright CD11b staining and coinciding with intense I-A and positive CD11c staining were also found in the perivascular regions (Fig. 7⇓C), 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. 7⇓Cc, arrow).

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FIGURE 7.

Confocal microscopy of αE-DC and CD11b^high-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 CD11b^high-DC (yellow to white image). D and E, Anti-I-A and anti-β₇ 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 ×40 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.

αE-DC expresses the epidermal DC marker Langerin

To support the epithelial nature of αE-DC, an Affymetrix microarray database obtained from mRNA of sorted αE-DC and CD11b^high-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 αE-DC, and its expression is 12-fold higher in αE-DC than in CD11b^high-DC (Table II⇓). The microarray data were confirmed by real-time PCR, which also showed that αE-DC expressed much higher (23-fold) Langerin mRNA than CD11b^high-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 αE-DC, which showed bright I-A and integrin β₇ staining and are localized mostly either in the epithelium or arteriolar wall (Fig. 7⇑D). The results confirmed that αE-DC expressed Langerin and that it was the predominant cell type in lungs for this expression. The Langerin expression of αE-DC suggests that they are similar to Langerhans cells, CD8⁺ lymphoid DC, and DEC-205^highCD8^low lymph node DC, all of which express this protein (50, 51).

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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 αE-DC (lanes a) or CD11b^high-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 β-actin, 181 bp. Lanes 1 and 2 contained no cDNA. This analysis was performed twice.

αE-DC express Claudin-1, Caludin-7, and ZO-2

To gain access to Ag or pathogens in the airways, αE-DC in the mucosa must traverse the bronchial epithelial layer through the tight junction barrier. This is achieved by the αE-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 αE-DC compared with CD11b^high-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, αE-DC and CD11b^high-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 αE-DC expressed much higher levels of Claudin-1 and Claudin-7 and somewhat higher level of ZO-2 than CD11b^high-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 αE-DC (Claudin-1, Fig. 7⇑E; Claudin-7 is specifically expressed in αE-DC but is dim and, therefore, not shown) and the staining is colocalized with I-A and β₇ in the epithelia or vascular walls. These results showed that αE-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 αE-DC accumulation and activation in mice with Ag-induced inflammation

The role of lung αE-DC in asthma was examined in an OVA-induced asthma-like model (33). Immunized mice exhibited marked airway hyperresponsiveness and eosinophilia. αE-DC increased markedly, from an average of 1.9 × 10⁵ in control mice to 5.4 × 10⁵ αE-DC per lung in immunized mice. They constituted 21% of the total lung I-A^highCD11c^high-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 × 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 αE. In OVA-immunized mice, αE-negative lung DC increased by at least 12-fold compared with controls. Thus exogenous DC constituted at the most 0.7% of the αE-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. αE-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 αE-DC. However, the increase did not reach statistical significance because of the high scattering of the results from replicate experiments.

Large numbers of αE-DC and CD11b^high-DC were present in the mucosa of inflamed lungs

Confocal microscopy showed that as in control lungs, large numbers of αE-DC and CD11b^high-DC were present in the airway mucosa, adjacent to the vascular walls, and within the leukocyte infiltration areas (Fig. 7⇑B and 7⇑C, d–f). Many more CD11b^high-DC were found (Fig. 7⇑C), in agreement with the increases in lung CD11b^high-DC numbers (12-fold higher) in immunized mice. Interestingly, the preferential localization of αE-DC and CD11b^high-DC were different. αE-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 α_Eβ₇⁺ (Fig. 7⇑B). CD11b^high-DC, in contrast, were found in the proximal subepithelia and vascular wall as a minor I-A⁺CD11c⁺ population (Fig. 7⇑C, e and f). They occurred primarily interspersed within the leukocyte infiltration zone of the peribronchial and perivascular cuffs (Fig. 7⇑Cf, inset, arrows). The results suggest that αE-DC and CD11b^high-DC exhibit different preferential localization.