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The α1β1 and αEβ7 Integrins Define a Subset
of Dendritic Cells in Peripheral Lymph Nodes
with Unique Adhesive and Antigen Uptake
Properties
Jonathan T. Pribila, Andrea A. Itano, Kristen L. Mueller and
Yoji Shimizu
J Immunol 2004; 172:282-291; ;
doi: 10.4049/jimmunol.172.1.282
http://www.jimmunol.org/content/172/1/282
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Copyright © 2004 by The American Association of
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References
The Journal of Immunology
The ␣1␤1 and ␣E␤7 Integrins Define a Subset of Dendritic
Cells in Peripheral Lymph Nodes with Unique Adhesive and
Antigen Uptake Properties1
Jonathan T. Pribila,* Andrea A. Itano,† Kristen L. Mueller,* and Yoji Shimizu2*
D
endritic cells (DCs)3 are potent APCs that regulate the
immune response through the induction of adaptive immunity and the maintenance of peripheral tolerance (1,
2). As a population, DCs are composed of a number of functionally
and phenotypically distinct subsets that have been defined by differential expression of several cell surface receptors. Among the
most informative markers of DC subsets are integrins, a family of
␣␤ heterodimeric transmembrane glycoproteins that mediate adhesion to the extracellular matrix (ECM) as well as to other cells
(3). CD11c, the ␣X subunit of the ␣X␤2 integrin, was the first
murine DC marker to be identified (4) and has been instrumental
in the identification and characterization of DC subsets in the
lymph node (LN), spleen, and thymus (5–7). In addition to CD11c,
CD11b, the ␣M subunit of the ␣M␤2 (MAC-1) integrin, has become important in differentiating DC subsets (5). Originally identified as a myeloid-specific Ag, CD11b distinguishes myeloid DC
lacking CD8␣ expression from the lymphoid DC subset that expresses CD8␣ (5). Finally, recent evidence suggests that the ␣4
Departments of *Laboratory Medicine and Pathology and †Microbiology, Center for
Immunology, Cancer Center, University of Minnesota Medical School, Minneapolis,
MN 55455
Received for publication July 29, 2003. Accepted for publication October 23, 2003.
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 by National Institutes of Health Grants AI38474 and
AI31126 (to Y.S.), the Harry Kay Chair in Biomedical Research (to Y.S), a University
of Minnesota Doctoral Dissertation Fellowship (to J.T.P.), the Irvington Institute of
Immunological Research (to A.A.I.), and National Institutes of Health Training Grant
AI007313 (to K.L.M.).
2
Address correspondence and reprint requests to Dr. Yoji Shimizu, Department of
Laboratory Medicine and Pathology, University of Minnesota Medical School, MMC
334/Room 6-112 Basic Sciences and Biomedical Engineering Building, Minneapolis,
MN 55455. E-mail address: [email protected]
3
Abbreviations used in this paper: DC, dendritic cell; ECM, extracellular matrix;
HEV, high endothelial venule; HSA, human serum albumin; int, intermediate; LN,
lymph node; pLN, peripheral LN; RFP, red fluorescent protein; SA, streptavidin.
Copyright © 2004 by The American Association of Immunologists, Inc.
integrin subunit (CD49d) is a maturation marker on monocytederived human DCs (8).
In addition to their role as antigenic markers, integrins most
likely regulate DC function and localization in lymphoid tissue.
The LN is a highly structured tissue in which functional and spatial
separation is maintained by a reticular network of collagen fibers
and other ECM proteins (9). This network of conduits connects the
lymphatic vessels draining into the subcapsular sinus of LNs with
high endothelial venules (HEVs) and is critical for the movement
of soluble Ag and inflammatory cytokines from tissues into draining LNs (10). Although these fibers are ensheathed by fibroblastic
reticular cells, ⬃10% of the ECM fibers remain exposed to the
cellular compartment of the LN and may serve as potential binding
sites for DCs and other cell types (11). Within the LN, differences
in either the ECM content of the reticular network or the integrin
ligands produced by fibroblastic reticular cells that surround these
fibers may define discrete functional areas by specifically retaining
DCs expressing the appropriate integrins within those areas. The
ability of different DC subsets to interact with collagen and other
ECM proteins found in these fibers may be critical to our understanding of the function of these DC subsets, as recent studies have
shown that the uptake of soluble Ag by skin-derived DCs resident
in draining LNs is critical to the initial activation of naive T cells
(12). Because this network severely limits the diffusion of soluble
Ags in LNs (10), this suggests that DCs capable of adhering to
these ECM fibers are uniquely situated to serve as the critical
APCs that initiate T cell activation by acquiring soluble Ag and
presenting it to T cells. In situ analysis has revealed that DC subsets differentially expressing the ␣M integrin (CD11b) localize to
distinct areas of the LN (13), and the specific localization pattern
of each DC subset may be important to its role in the regulation of
the immune response (14, 15). However, the mechanism by which
DC subsets are situated in distinct regions of LNs, and particularly
the role of ECM-binding integrins in this process, is not known.
Integrins may also regulate DC function by controlling the ability of DCs to traffic to the LN from peripheral sites. Blood-borne
0022-1767/04/$02.00
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Dendritic cells (DCs) are a heterogeneous population of APCs with critical roles in T cell activation and immune regulation. We
report in this study the identification and characterization of a novel subset of DCs resident in skin-draining peripheral lymph
nodes of normal mice. This subset of CD11chighCD40highCD8␣intermediate (int) DCs expresses the collagen-binding integrin, ␣1␤1,
and the E-cadherin-binding integrin, ␣E␤7. Although ␣1␤1 and ␣E␤7 are also expressed on CD11chighCD40intCD8␣high lymphoid
DCs, CD11chighCD40highCD8␣int DCs demonstrate preferential integrin-mediated adhesion to collagen and fibronectin. This DC
subset most likely acquires expression of these integrins in peripheral lymph node, as this subset is not found in the spleen or
mesenteric lymph node, and recent DC migrants from the skin lack expression of ␣1␤1 and ␣E␤7 integrins. Resident CD40high DCs
express ␣1␤1 integrin and colocalize with collagen in lymph nodes. When compared with CD11chighCD40highCD8␣int DCs lacking
expression of these integrins, the ␣1␤1ⴙ␣E␤7ⴙ DC subset exhibits more efficient formation of Ag-independent conjugates with T
cells, and a decreased ability to acquire soluble Ag. Thus, the ␣1␤1 and ␣E␤7 integrins define a unique population of peripheral
lymph node-derived DCs with altered functional properties and adhesive potential that localizes these cells to sites in lymph nodes
where Ag presentation to T cells occurs. The Journal of Immunology, 2004, 172: 282–291.
The Journal of Immunology
DCs enter the LN through the HEVs, and both ␤1 and ␤2 integrins
mediate adhesion of blood-borne DCs to human umbilical vein
endothelium (16). By contrast, DCs migrating from solid tissue
such as skin arrive at the LN via the afferent lymph. Contact hypersensitivity models have clearly demonstrated that the ␣L␤2
(LFA-1) integrin and its ligand, ICAM-1, are required for the migration of Langerhans cells to the LN (17, 18). Similar studies
have also demonstrated a role for ␣6 integrins, which mediate adhesion to the ECM protein laminin, in Langerhans cell migration
out of the skin (19). In addition to the role of integrins in DC
homing to the LN, ␤2 integrins are critical for the homeostatic
accumulation of DC within the lungs (20).
In this study, we identify and characterize a novel subpopulation
of DCs in normal mouse peripheral LNs (pLNs) that coordinately
expresses high levels of the ␣1␤1 and ␣E␤7 integrins and exhibits
unique adhesive and Ag uptake capabilities.
Materials and Methods
Mice
DC isolation
DCs were isolated from pLN, as described (13). Briefly, superficial inguinal, axillary, superficial cervical, and mandibular LN were isolated from at
least two mice and teased apart in a 400 U/ml collagenase D (Roche Molecular Biochemicals, Indianapolis, IN) solution in RPMI 1640 supplemented with 10 mM HEPES, pH 7.2, 2% FCS, penicillin/streptomycin, and
L-glutamine. LNs were incubated at 37°C for 20 min before stopping the
digestion with 0.1 M EDTA solution (pH 7.2). The LNs were filtered and
washed twice in PBS. In experiments in which mesenteric LNs were used,
they were treated in the same manner as the pLNs.
tinylated goat anti-hamster Ab. Unbound secondary Abs were washed off,
and any Ag-binding epitopes were blocked with hamster IgG. Finally, the
cells were stained with a mixture of Abs that included CD11c PE, CD40
PE/Cy5, ␣E FITC M290, and SA-APC, and analyzed, as described above.
Adhesion assay
Flat-bottom 96-well microtiter plates (Costar, Cambridge, MA) were
coated overnight at 4°C with the indicated amount of one of the following
integrin substrates: type IV collagen (Life Technologies, Rockville, MD),
type III collagen (Fibrogen, South San Francisco, CA), laminin (Life Technologies), E-cadherin (R&D Systems, Minneapolis, MN), human plasma
fibronectin (Invitrogen Life Technologies, Carlsbad, CA), or murine fibronectin (Invitrogen). Unbound sites were blocked with 0.5% human serum albumin (HSA) for 2 h at 37°C. Three hundred thousand cells were
added per well in a final volume of 0.1 ml PBS containing 0.5% HSA. The
cells were allowed to settle on the plate for 60 min at 4°C. Following 20
min of stimulation at 37°C, the nonadherent cells were washed away and
the adherent cells were removed using a 0.1 mM EDTA solution. For each
condition, duplicate samples of cells pooled from six wells were washed in
FACS buffer. Samples were stained with CD8␣ FITC, CD40 PE/Cy5, and
CD11c PE (as described above), washed, and then resuspended in exactly
200 ␮l FACS buffer plus 50 ␮l PKH26 microbeads (Sigma-Aldrich, St.
Louis, MO) for a total sample volume of 250 ␮l. Each sample was collected on a FACScan (BD Biosciences) for ⬃4 min and analyzed using
CellQuest software (BD Biosciences). Triplicate samples of preadherent
cell populations were prepared in a similar manner, and all of the cells were
analyzed by flow cytometry. The number of DCs, based on differential
expression of CD8␣ and CD40 expression on the CD11chigh cells, in each
sample was determined, as previously described (24).
In vivo skin sensitization
Migration of skin-derived DCs to the draining LN was determined, as
previously described (7, 19). Green fluorescent Cell Tracker (Molecular
Probes, Eugene, OR) was resuspended at a concentration of 10 mM in
DMSO. Just before application, the Cell Tracker dye was diluted to a final
concentration of 3.3 mM in a 50/50 (v/v) acetone/dibutyl phthalate mixture. Mice were painted with 50 ␮l on both the dorsal and ventral sides of
the ears. After 18 h, LNs were harvested and the DCs were collected and
phenotyped, as described above. Briefly, cells were stained with unconjugated anti-␣1 or anti-␣E integrin Abs, followed by biotinylated secondary
mAb. Unbound epitopes on the secondary Abs were blocked, and the cells
were stained with CD11c PE, CD40 PE/Cy5, and SA-APC. Cells were
analyzed, as described above.
Abs and reagents
DC subsets were identified by staining with anti-CD11c PE or FITC mAb
HL3 (BD PharMingen, San Diego, CA), and anti-CD8␣ FITC and antiCD40 PE/Cy5 mAb 1C10 (both from eBioscience, San Diego, CA). Integrin expression was determined using Abs against integrin ␣1 (CD49a)
(Ha31/8), ␣2 (CD49b) (Ha1/29), ␣4 (CD49d) (R1-2), ␣5 (CD49e) (5H10-27 (MFR5)), ␣L (CD11a) (M17/4), ␤1 (CD29) (HM␤1-1), ␣E (CD103)
(M290), or ␣E FITC (M290), and ␤4 (CD104) (346-11a), all of which were
purchased from BD PharMingen. Abs against ␣3 (CD49c) were purchased
from BD Transduction Laboratories (San Diego, CA). Integrin ␣6 (CD49f)
was stained using Hm␣6 mAb (kindly provided by H. Yagita, Juntendo
University, Tokyo, Japan) (22, 23).
Flow cytometry
To characterize integrin expression on DC subsets, DCs were isolated, as
described above, and resuspended in FACS buffer (HBSS supplemented
with 1% BCS and 0.2% sodium azide). Fc receptors were blocked for 15
min at 4°C with either anti-FcR Ab (clone 2.4G2) or mouse IgG and then
stained with unconjugated Abs against specific integrin subunits for 30 min
at 4°C. Excess Ab was washed off, and the cells were stained with either
biotinylated goat anti-hamster or biotinylated goat anti-rat depending on
the primary Ab. Unbound secondary Abs were washed off, and any free
epitopes were blocked with hamster IgG or rat IgG. Finally, the cells were
stained with a mixture of CD8␣ FITC, CD11c PE, CD40 PE/Cy5, and
streptavidin APC (SA-APC). In each experiment, ⬎750,000 cells for each
sample were acquired with a FACSCalibur (BD Biosciences, San Jose,
CA) and subsequently analyzed using CellQuest software (BD Biosciences). The DCs were identified by gating on the CD11chigh cells. This
population of cells was then subdivided based on CD8␣ and CD40 expression (Fig. 1A).
To determine expression of multiple integrins on DCs, Fc receptors
were blocked, as described above, and the cells were subsequently stained
with the anti-␣1 mAb Ha31/8 for 30 min at 4°C and followed by a bio-
Immunofluorescent microscopy
pLN tissue sections were prepared and stained, as previously described
(12). Detection of integrin ␣1 or ␣2 chain colocalization with collagen was
performed using either hamster anti-mouse ␣1 integrin or hamster antimouse ␣2 integrin Abs, followed by biotinylated goat anti-hamster IgG
(Caltag, Burlingame, CA), SA-conjugated HRP (SA-HRP), and tyramide
FITC (NEN, Boston, MA) or tyramide rhodamine (NEN). Collagen was
detected using a biotinylated goat anti-collagen type III Ab (Southern Biotechnology Associates, Birmingham, AL), followed by SA-HRP and tyramide Cy5 (NEN). Integrin ␣1 colocalization with CD40 was detected with
hamster anti-mouse ␣1 integrin (as described above) and biotinylated rat
anti-mouse CD40 (Caltag), followed by SA-HRP, biotinyl tyramide
(NEN), and SA-Cy5 (Caltag). Images were acquired using a Bio-Rad
MRC-1000 confocal microscope equipped with a krypton/argon laser (BioRad Life Sciences, Hercules, CA) and CoMOS v. 7.0a (Bio-Rad Life Sciences) software. Separate green, red, or far red (Cy5) images were collected for each section analyzed. Final image processing was performed
using Photoshop software (Adobe, San Jose, CA).
Conjugate assay
pLNs and mesenteric LNs were isolated from OT-I TCR transgenic mice
that express the CD90 isoform, Thy-1.1. T cells were isolated by negative
selection using MACS magnetic separation technology (Miltenyi Biotec,
Auburn, CA). Briefly, LN cells were incubated in a mixture of the following Abs for 20 min at 4°C: goat anti-mouse IgG FITC (0.5 ␮g/106 cells)
(Southern Biotechnology Associates), anti-B220 FITC (0.07 ␮g/106 cells)
(eBioscience), anti-I-Ab FITC (0.07 ␮g/106 cells) (BD PharMingen), and
anti-␣E FITC (0.07 ␮g/106 cells), followed by incubation with anti-FITC
microbeads for 20 min at 4°C (10 ␮l beads/10 ⫻ 106 cells). Cells were
washed twice and run over MS or LS separation columns.
For conjugate assays, DCs from normal, nontransgenic B6 mice were
isolated, as described above, and resuspended at 20 ⫻ 106 cells/ml in
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C57BL/6 (B6) mice were purchased from the National Cancer Institute
(Bethesda, MD) and were housed in specific pathogen-free isolation rooms
at the University of Minnesota (Minneapolis, MN). OT-I TCR transgenic
mice on a B6/PL background were provided by K. Hogquist (University of
Minnesota) (21). Mice were used between 8 and 12 wk of age and housed
in specific pathogen-free facilities at the University of Minnesota. All experimental protocols involving the use of mice were approved by the Institutional Animal Care and Use Committee at the University of Minnesota.
283
284
Ag uptake
The ears of C57BL/6 mice were injected s.c. with 50 ␮g of red fluorescent
protein DsRed (E␣RFP), as described (12). Four hours following injection,
DCs were isolated, as described above, from the draining and nondraining
LNs. DCs were stained, as described above, with unconjugated anti-␣1
integrin Ab, followed by biotinylated secondary mAb. Unbound epitopes
on the secondary Abs were blocked, and the cells were then stained with
anti-CD11c FITC, anti-CD40 PE/Cy5, and SA-APC. CD11chighCD40high
DCs were identified and analyzed, as described above.
Statistical analysis
Statistical analysis was performed using the Student’s t test.
Results
Identification of DC subsets
To minimize effects of ex vivo purification on DC phenotype, we used
multicolor flow cytometry to differentiate DC subsets expressing
CD11c, CD40, and CD8␣. Using these markers, we identified three
populations of CD11chigh DCs in pLN: CD40intermediate (int)CD8␣low
(24%), CD40intCD8␣high (25%), and CD40highCD8␣int (18%) (Fig.
1A). Consistent with previous results (7), the CD40intCD8␣low and the
CD40intCD8␣high populations were also found in the mesenteric LNs
and spleen. Classically, these two DC subsets have been designated as myeloid (CD11chighCD40intCD8␣low) and lymphoid
(CD11chighCD40intCD8␣high) DCs (25, 26). Although it is clear that
these designations no longer reflect the developmental lineage of
these cells (27–29), we will use these terms to describe DCs with the
aforementioned phenotypes. In contrast to myeloid and lymphoid
DCs, a population of CD11chighCD40highCD8␣int DCs was found
exclusively within skin-draining pLN. Although DCs from the skin
could be of epidermal and dermal origin, CD11c and CD8␣ have been
reported to be differentially expressed by these populations. The
expression of high levels of CD11c and intermediate levels of CD8␣
is consistent with the phenotype of Langerhans cells (7).
FIGURE 1. Integrin expression on CD11chigh DC subsets in the pLN. LN cells from normal B6 mice were stained with CD8␣ FITC, CD11c PE, and
CD40 PE/Cy5, as described in Materials and Methods. A, CD40 and CD8␣ expression on CD11chigh cells (based on the gate shown on the left contour
plot) from pLN, mesenteric LNs (mLN), and spleens of B6 mice. B, Using the gates shown in A, four-color flow cytometry was used to assess the expression
of integrin ␣ and ␤ subunits on CD11chigh DC subsets in pLN. Filled histograms show integrin expression on pLN-derived DCs (CD40highCD8int), myeloid
DCs (CD40intCD8␣low), and lymphoid DCs (CD40intCD8␣high). The negative controls for each DC subset are shown with empty histograms. Data are
representative of at least three independent experiments.
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RPMI 1640 supplemented with 10% FCS (RP10). DCs were pulsed with 1
␮M or 100 nM OVA peptide (SIINFEKL), 1 ␮M SIY peptide
(SIYRYYGL), or no peptide for 30 min at 37°C. Peptides were synthesized
by Research Genetics (Carlsbad, CA). After pulsing, cells were washed and
resuspended in ice-cold RP10. DCs and T cells were then added to flatbottom 96-well microtiter plates at a concentration of 2 ⫻ 106 DCs/well
and 0.5 ⫻ 106 T cells/well in a total volume of 200 ␮l/well. Plates were
centrifuged for 1 min at 1000 rpm and then incubated for 30 min at 37°C.
Following conjugation, plates were placed on ice and Fc receptors were
blocked using anti-FcR Ab (clone 2.4G2) for 15 min. Cells were then
stained with anti-␣E FITC, anti-CD11c PE, anti-CD40 PE/Cy5, and antiThy-1.1 APC (eBioscience) Abs for 30 min. Plates were washed with 200
␮l FACS buffer, spun down, and fixed with 1% paraformaldehyde for 20
min at room temperature. Cells were washed twice with 200 ␮l FACS
buffer and harvested from the plate. For each condition, duplicate samples
of cells were pooled from three wells, and each sample was analyzed with
a FACSCalibur (BD Biosciences) using CellQuest software (BD Biosciences). To determine the degree of conjugate formation, we gated on the
cells expressing high levels of CD11c and CD40. Then using ␣E integrin
expression, this population was subdivided into two subpopulations:
CD11chighCD40high ␣E positive and CD11chighCD40high ␣E negative. The
percentage of conjugate formation with OT-I T cells was determined as the
number of cells within each subpopulation that exhibit FL-4 (Thy-1.1)
fluorescence divided by the total number of cells in each subpopulation.
DCs AND ECM-BINDING INTEGRINS
The Journal of Immunology
285
Integrins are differentially expressed on the CD11c subsets of
murine DCs
FIGURE 2. ␣1␤1 and ␣E␤7 integrin expression on DC subsets in the
spleen and mesenteric LN. Using the gates shown in Fig. 1A, ␣1 and ␣E
integrin expression was assessed on myeloid and lymphoid DCs fron
spleen and mesenteric LN (mLN). Filled histograms show integrin expression, and negative controls for each DC subset are shown with empty
histograms. Data are representative of at least three independent
experiments.
spleen or mesenteric LNs with low or intermediate CD8␣ expression that also expresses ␣1 and/or ␣E integrin.
␣1 and ␣E integrin are expressed on the same subset of
CD40highCD8␣int pLN-derived DCs
Among the three CD11chigh DC subsets in pLN that we analyzed
for integrin expression, we observed the most heterogeneity in
integrin expression on CD40highCD8␣int DCs. Specifically, ␣1␤1
and ␣E␤7 were expressed on only a subpopulation of
CD11chighCD40highCD8␣int cells (Fig. 1B). We performed additional flow cytometric studies to determine whether ␣E␤7 and ␣1␤1
were expressed on the same subset of CD11chighCD40high pLNderived DCs. Fig. 3 shows that all of the ␣E-positive
FIGURE 3. Coexpression of ␣E␤7 and ␣1␤1 integrin on the same subset of
pLN-derived CD11chighCD40high DC. pLN cells from B6 mice were stained with
anti-CD11c PE and anti-CD40 PE/Cy5. Top, Contour plot of ␣E integrin expression (x-axis) and ␣1 integrin expression (y-axis) on CD11chighCD40high DC. Bottom, Forward and side scatter properties of the CD11chighCD40highCD8␣int␣E⫺
DC (left panel) and CD11chighCD40highCD8␣int␣E⫹ DC (right panel). The mean
forward light scatter value was 633 for the ␣E-negative subset compared with 718
for the ␣E⫹ subset, and the mean side scatter value was 242 for the ␣E-negative
cells compared with 305 for the ␣E⫹ cells. Results are representative of at least
three independent experiments.
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Using these gates, we used four-color flow cytometry to examine
the expression of several integrin ␣ and ␤ subunits on each subset
of CD11chigh DCs in pLN. In addition to CD11c (␣X integrin
subunit), the expression of ␣M (CD11b, MAC-1) has been used to
differentiate the myeloid and lymphoid DC subsets. In agreement
with previous results (5, 30), we found that ␣M was highly expressed on the myeloid and CD40highCD8␣int pLN-derived DCs,
but was expressed at low levels on lymphoid DCs (Fig. 1B). In
contrast, the ␣L integrin subunit (CD11a) was uniformly expressed
on each of the CD11chigh DC subsets (Fig. 1B) (30).
In contrast to the ␤2 family of integrins, little is known about the
expression of the integrins ␤1 and ␤7 subunits, or the ␣ subunits
that pair with these integrin ␤-chains, on murine DC subsets. As
shown in Fig. 1B, the integrin ␤1 subunit was expressed on all
three CD11chigh DC subsets. The ␣4 and ␣5 subunits, which associate with ␤1 to form the fibronectin-binding integrins, ␣4␤1 and
␣5␤1, were also expressed at similar and uniform levels on each
CD11chigh DC subset. Although ␣5 dimerizes exclusively with ␤1,
␣4 may form heterodimers with either ␤1 or ␤7. Although the ␤7
integrin subunit was expressed on all three DC subsets, there was
a subpopulation of CD40highCD8␣int pLN-derived DCs that expressed high levels of ␤7 (Fig. 1B). Because both ␤1 and ␤7 are
expressed on all of the CD11chigh subsets, it is possible that DCs
express both ␣4␤1 and ␣4␤7 integrin.
In contrast to the fibronectin-binding integrins, several ␤1 integrin subunit-associating ␣ subunits are differentially expressed on
CD11chigh DC subsets. The ␣6 integrin subunit was expressed predominately by the lymphoid DC subset and to a lesser extent by
myeloid DCs. Consistent with previous reports, we did not detect
appreciable ␣6 integrin expression on CD40highCD8␣int pLN-derived DCs (19). Although the ␣6 integrin subunit can form two
different laminin-binding integrins, ␣6␤1 and ␣6␤4, the ␤4 subunit
was not expressed on any of the CD11chigh DC subsets (Fig. 1B).
This suggests that ␣6␤1 is the only ␣6-containing integrin expressed on CD11chigh DCs.
The expression of the ␣1 integrin subunit was restricted to particular subsets of CD11chigh cells. As shown in Fig. 1B, ␣1 was
uniformly expressed on lymphoid DCs, but was not expressed on
myeloid DCs. Thus, similar to CD8␣ and ␣M integrin, ␣1 integrin
is differentially expressed on these two DC subsets. Integrin ␣1
was also expressed on ⬃50% of the CD40highCD8␣int pLN-derived DCs, suggesting that there is heterogeneity within this DC
subset. In contrast, the ␣2 and ␣3 integrins, which can also pair
with ␤1, were not expressed on any of the CD11chigh DC subsets
(Figs. 1B).
The ␤7 integrin subunit can also pair with the ␣E integrin subunit to form the ␣E␤7 integrin, which mediates adhesion of intraepithelial lymphocytes to E-cadherin (31, 32). Like the ␣1 integrin
subunit, the ␣E integrin subunit was differentially expressed on the
CD40int DCs, with higher expression on the lymphoid DCs. In
addition, like ␣1, the ␣E integrin subunit was expressed on a subpopulation (⬃30%) of the CD40highCD8␣int pLN-derived DCs.
Thus, both the ␣1 integrin subunit and the ␣E integrin subunit
suggest heterogeneity within this subset of pLN-derived DCs.
We also examined ␣1 and ␣E integrin expression on CD11chigh
DCs found in spleen. Similar to the results obtained with skindraining pLN, ␣1 and ␣E integrin expression was detected on
splenic lymphoid DCs, but not on splenic myeloid DCs (Fig. 2).
Similar results were obtained when examining DCs isolated from
mesenteric LN (Fig. 2 and data not shown). Thus, unlike what was
observed in pLNs, we did not detect a subset of DCs in either the
286
DCs AND ECM-BINDING INTEGRINS
CD40highCD8␣int pLN-derived DCs also expressed high levels of
the ␣1 integrin. In contrast, the ␣E-negative CD40highCD8␣int
pLN-derived DCs could be further divided into subsets that expressed either no ␣1 integrin or low levels of ␣1 integrin. In addition, the forward and side scatter properties of CD40highCD8␣int
pLN-derived DCs differed based on expression of ␣E. The
CD11chighCD40high pLN-derived DCs that expressed high levels
of both ␣E and ␣1 were consistently larger and more granular than
the cells lacking ␣E, with increased mean forward scatter and mean
side scatter in the ␣E-positive CD40highCD8␣int cells compared
with the ␣E-negative CD40highCD8␣int cells. Thus, these results
demonstrate that there is a subset of CD40highCD8␣int pLN-derived DCs that is defined by high levels of expression of both the
␣1␤1 and ␣E␤7 integrins, and unique forward and side scatter
properties.
Localization of ␣1 and ␣2 integrins within the LN
Functional and spatial separation within the LN is maintained by a
reticular network of collagen and other ECM fibers ensheathed by
fibroblastic reticular cells. We used immunohistochemistry to determine the localization of ␣1 integrin-expressing cells within the
LN and their proximity to collagen filaments (Fig. 4). Cells expressing ␣1 integrin were located throughout the cortex of the LN,
and there was extensive colocalization of anti-␣1 integrin Ab staining with anti-type III collagen Ab staining. Both type III collagen
and ␣1 integrin-expressing cells were excluded from the follicles.
Because the ␣1␤1 integrin mediates cell adhesion to collagen (33),
the extensive overlap of ␣1 integrin and collagen throughout the
LN suggests that fibroblastic reticular cells may be expressing
␣1␤1 integrin. In contrast, cells expressing another collagen-binding integrin, ␣2␤1, localized on the lumenal side of structures that
are consistent with HEVs (Fig. 4). The lack of ␣2 integrin-expressing cells outside of HEVs is consistent with the lack of ␣2 integrin
expression on any of the DC subsets that we examined by flow
cytometry (Fig. 1B).
To identify DCs within the LN, sections were stained with an
anti-CD40 Ab. Although there are many cells in the LN that express moderate levels of CD40 (7), we were only able to detect the
cells expressing the highest levels of CD40, as demonstrated by the
lack of appreciable anti-CD40 staining in follicles. LN sections
stained with both anti-␣1 and anti-CD40 clearly demonstrated that
some, but not all, of the CD40high cells also expressed ␣1 integrin
(Fig. 4). These data are consistent with expression of ␣1 on a
subset of CD40highCD8␣int pLN-derived DCs (Fig. 1B).
CD11chigh DC subsets adhere to fibronectin in an integrindependent manner
We next used in vitro adhesion assays to assess the adhesion of
CD11chigh DC subsets to ␤1 integrin ligands. Each of the
CD11chigh DC subsets adhered to human and mouse fibronectin in
a dose-dependent manner (Fig. 5A and data not shown). We consistently observed slightly higher levels of adhesion of
CD40highCD8␣int pLN-derived DCs to fibronectin when compared
with either the myeloid or lymphoid DC subsets. Ab-blocking
studies demonstrated that an anti-␤1 integrin Ab completely reduced adhesion of all three DC subsets to background levels (Fig.
5B). Adhesion of CD40highCD8␣int pLN-derived DCs to fibronectin was partially blocked by an anti-␣4 or an anti-␣5 Ab, suggesting
a role for both ␣4 and ␣5 integrins in mediating adhesion of these
DCs to fibronectin. In contrast, adhesion of myeloid and lymphoid
DCs to fibronectin was only blocked by an anti-␣4 Ab, while the
anti-␣5 Ab had a minimal effect, even when used in combination
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FIGURE 4. Cells expressing ␣1 integrin colocalize with collagen in pLN. Fixed and frozen sections of pLN were stained, as described in Materials and
Methods, with Abs specific for type III collagen and ␣2 integrin (left panels), type III collagen and ␣1 integrin (middle panels), or CD40 and ␣1 integrin
(right panels). The top set of panels shows pLN sections at a ⫻10 magnification, and the bottom set of panels shows pLN sections at a ⫻20 magnification.
Note that cells expressing ␣1 integrins colocalize extensively with collagen throughout the cortex of the pLN. A portion of these ␣1-expressing cells also
expresses high levels of CD40. Cells expressing ␣2 are localized exclusively to the lumenal side of HEVs. Follicles are marked with F.
The Journal of Immunology
287
␣L integrin Ab had no effect on adhesion of this subset of DCs to
collagen. CD40highCD8␣int pLN-derived DCs also adhered preferentially to a recombinant form of human type III collagen, although overall levels of adhesion to type III collagen were slightly
lower than that observed with type IV collagen (data not shown).
Because both a subset of CD40highCD8␣int pLN-derived DCs
and lymphoid DCs express the ␣E and ␤7 integrins, we examined
adhesion of DCs to purified E-cadherin. Although lymphoid DCs
express ␣E␤7, we did not detect adhesion of these cells to E-cadherin above background (Fig. 6C). As expected, myeloid DCs,
which do not express ␣E␤7, also did not adhere to E-cadherin. By
contrast, the CD40highCD8␣int pLN-derived DCs adhered to Ecadherin, with optimal adhesion at a concentration of 0.1 ␮g/well
of E-cadherin.
Langerhans cells in the skin and DC migrants from the skin do
not express ␣1 and ␣E integrins
with the anti-␣4 Ab (Fig. 5B). Thus, even though ␣5␤1 integrin is
expressed on both myeloid and lymphoid DCs, we could not detect
appreciable ␣5␤1-dependent adhesion of these DCs to fibronectin.
Adhesion of the three DC subsets to fibronectin was unaffected by
an inhibitory anti-␣L Ab.
CD40highCD8␣int pLN-derived DCs, but not other DC subsets,
adhere to collagen and E-cadherin
Because ␣1␤1 integrin is differentially expressed on CD11chigh
DCs, we also analyzed the adhesion of DCs to murine type IV
collagen. As shown in Fig. 6A, neither myeloid nor lymphoid DCs
adhered to collagen in a dose-dependent manner, with adhesion
ranging between 5 and 13% above background at all of the doses
tested. This result was somewhat surprising, because lymphoid
DCs express ␣1␤1 integrin (Fig. 1B). In contrast to the CD40int
DCs, CD40highCD8␣int pLN-derived DCs adhered to collagen in a
dose-dependent manner, with maximal adhesion of 30% over
background at the highest dose of collagen tested (Fig. 6A). This is
consistent with the expression of ␣1␤1 integrin on a subset of these
DCs. Adhesion of CD40highCD8␣int pLN-derived DCs to type IV
collagen was mediated, in part, by ␣1␤1 integrin, because blocking
Abs against either the ␣1 or ␤1 subunit reduced adhesion to collagen compared with untreated cells (Fig. 6B). In contrast, an anti-
Conjugate formation with Ag-specific T cells
Using ␣E integrin as a marker to distinguish subsets of
CD11chighCD40high pLN-derived DCs, we examined the ability of
these DC subsets to form stable conjugates with OT-I TCR transgenic T cells. Purified OT-I T cells were allowed to interact with
pLN cells, and multicolor flow cytometry was used to enumerate
the percentage of ␣E-positive and ␣E-negative CD11chighCD40high
DCs in conjugates with T cells. In the absence of relevant peptide
Ag, a low, but detectable, percentage of CD11chighCD40high pLNderived DCs formed conjugates with OT-I T cells (Fig. 8A). However, we consistently noted that the percentage of ␣E-positive
CD11chighCD40high DCs in conjugates with OT-I T cells was 1.5to 2-fold higher than the percentage of ␣E-negative
CD11chighCD40high DCs in conjugates. If DCs were first pulsed
with peptide Ag (OVA peptide SIINFEKL) before conjugate formation, the percentage of both ␣E-positive and ␣E-negative skinderived DCs in conjugates with T cells dramatically increased. We
also observed higher levels of Ag-dependent conjugates between
␣E-positive pLN-derived DCs and OT-I T cells compared with
␣E-negative pLN-derived DCs (Fig. 8A).
Acquisition of soluble Ag by resident skin-derived DCs in the LN
Itano et al. (12) have recently used a fluorescent Ag to show that
skin-derived DCs resident in lymph nodes can acquire s.c. injected
soluble Ag and initiate T cell activation. Using this system, we
assessed potential differences in the ability of subsets of pLNderived DC defined by differential ␣1 integrin expression to acquire this Ag, which consists of a portion of the I-Ed␣ MHC class
II subunit fused to the E␣RFP. As previously demonstrated, Ag
administered s.c. was present exclusively in the draining LN (Fig.
8B), and there was minimal Ag uptake by myeloid and lymphoid
DCs 4 h after Ag administration (data not shown). Ag uptake by
CD11chighCD40high pLN-derived DCs was detected. Interestingly,
there was an inverse relationship between ␣1 integrin expression
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FIGURE 5. CD11chigh DC subsets adhere to fibronectin in an integrindependent manner. A, Adhesion of CD40highCD8␣int pLN-derived DCs
(circles), myeloid DCs (squares), and lymphoid DCs (triangles) from pLN
to increasing amounts of fibronectin was assessed, as described in Materials and Methods. B, Adhesion of DC subsets to 1.5 ␮g/well of human
fibronectin was assessed in the absence of Ab (untreated) or in the presence
of Abs specific for the ␣L integrin (clone M17/4), ␣4 integrin (clone R1-2),
␣5 integrin (clone 5H10-27), ␤1 integrin (clone Ha2/5), or a combination of
anti-␣4 and anti-␣5 integrin Abs. In each experiment, the mean percentage
of adhesion of duplicate samples, each consisting of six pooled wells ⫾
SD, is shown. Background adhesion of DC subsets to HSA (⬍22%) was
subtracted. When compared with adhesion to HSA, the adhesion of all DC
subsets to fibronectin was statistically different (p ⬍ 0.01). ⴱ, p ⬍ 0.01; ⴱⴱ,
p ⬍ 0.02. Results are representative of at least three independent
experiments.
Given the heterogeneity of ␣1␤1 and ␣E␤7 expression on
CD40highCD8␣int pLN-derived DCs in the LN, we used immunohistochemistry to determine integrin expression on Langerhans
cells in the epidermis. We were unable to detect any ␣1- or
␣E-expressing epidermal DCs (data not shown). To confirm these
results, we applied Cell Tracker dye to the ears of mice and assessed
integrin expression on CD11chighCD40high DCs in pLN that had
recently migrated from the skin. Although a subpopulation of Cell
Tracker-negative CD11chighCD40high cells expressed both ␣1 and ␣E,
CD11chighCD40high cells that were labeled with Cell Tracker were
uniformly low in expression of both ␣1 and ␣E integrin (Fig. 7).
288
DCs AND ECM-BINDING INTEGRINS
on CD11chighCD40high pLN-derived DCs and Ag uptake. We observed that higher levels of E␣RFP were detected in the ␣1-negative CD11chighCD40high pLN-derived DC subset when compared
with E␣RFP uptake by ␣1-positive CD11chighCD40high pLN-derived DCs (Fig. 8B). Thus, these studies suggest differences in
efficiency of Ag uptake by DC subsets defined by differential expression of the ␣1 integrin.
Discussion
In this study, we demonstrate that ␤1 and ␤7 integrins are differentially expressed on CD11chigh DC subsets in the pLN of normal
mice. Although the expression of fibronectin-binding integrins and
adhesion to fibronectin are common to all DC subsets, we show
that ␣1␤1 and ␣E␤7 integrins are important phenotypic and functional DC markers. Specifically, we describe a subpopulation of
CD40highCD8␣int pLN-derived DCs that coordinately expresses
the ␣1␤1 and ␣E␤7 integrins. When compared with other
CD11chighCD40high pLN-derived DCs, this subpopulation has a
number of unique properties, including: 1) increased size and granularity; 2) ability to adhere to collagen and E-cadherin in vitro; 3)
enhanced ability to form Ag-independent conjugates with T cells;
and 4) decreased efficiency of acquisition of a soluble s.c. Ag.
CD40high DCs expressing ␣1␤1 integrin colocalize with collagen
in LN, but ␣1 and ␣E are not expressed on CD11chigh cells in the
skin and are not present on CD11chighCD40high cells that have
recently emigrated from the skin. Although the ␣X (CD11c) and
␣M (CD11b) integrin subunits are routinely used as DC markers
(1), analysis of the expression and function of other integrin subunits critical for cell adhesion and migration is lacking. Phenotypic
analysis clearly shows that the CD11chighCD40highCD8␣int DCs
found in normal mouse pLN can be subdivided into two distinct
subpopulations defined by differential expression of both ␣1␤1 and
␣E␤7 integrin. Differential expression of ␣1␤1 integrin is particularly intriguing, as ␣1␤1 interacts with collagen (33), an ECM protein that is a primary component of the reticular network that forms
a major structural scaffold in the LN (9). Besides the fibroblastic
reticular cells that ensheath these collagen-laden reticular fibers,
little is known about the other cells in the LN that interact with
these fibers. Our results suggest that, among the DC subsets examined, CD11chighCD40highCD8␣int pLN-derived DCs have enhanced capacity to interact with collagen found in the reticular
network, because these DCs preferentially adhere to collagen in
vitro. This suggests that this DC subset may be uniquely situated
to acquire soluble Ag and immunoregulatory cytokines arriving
from tissue sites via the afferent lymph, because these low m.w.
molecules percolate into LN via the reticular network (10, 12). In
addition to localizing DCs within the LN, collagen may directly
regulate DC function by promoting secretion of IL-12 and the
generation of a Th1 response (34).
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FIGURE 6. CD11chighCD40high pLN-derived DCs adhere to type IV collagen and E-cadherin. A, Adhesion of CD40highCD8␣int pLN-derived DCs
(circles), myeloid DCs (squares), and lymphoid DCs (triangles) from pLNs of B6 mice to increasing amounts of type IV collagen was assessed, as described
in Materials and Methods. ⴱ, p ⬍ 0.01 when compared with adhesion to HSA. B, Adhesion of CD40highCD8␣int pLN-derived DCs to 1.5 ␮g/well of
collagen was determined either in the absence of Ab (untreated) or in the presence of 10 ␮g/ml of Abs specific for ␣L integrin (clone M174), ␤1 integrin
(clone Ha2/5), or ␣1 integrin (clone Ha31/8). ⴱ, p ⬍ 0.01 when compared with adhesion to HSA; ⴱⴱ, p ⬍ 0.08 when compared with adhesion of untreated
CD40highCD8␣int pLN-derived DCs; ⴱⴱⴱ, p ⬍ 0.02 when compared with adhesion of untreated CD40highCD8␣int pLN-derived DCs. C, CD11chighCD40high
pLN-derived DCs (f), myeloid DCs (u), and lymphoid DCs (f) were isolated from pLN, and adhesion to 0.1 ␮g/well of E-cadherin was assessed using
the in vitro adhesion assay described in Materials and Methods. ⴱ, p ⬍ 0.10 of adhesion of CD40highCD8␣int pLN-derived DCs to E-cadherin when
compared with adhesion to HSA; ⴱⴱ, p ⬍ 0.02 of adhesion of CD40highCD8␣int pLN-derived DCs to E-cadherin when compared with adhesion of myeloid
DCs to E-cadherin; ⴱⴱⴱ, p ⬍ 0.07 of adhesion of CD40highCD8␣int pLN-derived DCs to E-cadherin when compared with adhesion of lymphoid DCs to
E-cadherin. In each experiment, the mean percentage of duplicate samples, each consisting of six pooled wells ⫾ SD, is shown. Background adhesion to
HSA (⬍22%) was subtracted. Results are representative of at least three independent experiments.
The Journal of Immunology
289
Immunohistochemistry analysis demonstrated the colocalization
of CD40high DCs expressing ␣1␤1 integrins with collagen in lymph
nodes in vivo. However, it is likely that both ␣1␤1-positive and
␣1␤1-negative CD40highCD8␣int pLN-derived DCs interact with
the collagen-rich reticular network in vivo, because CD40high DCs
lacking expression of ␣1␤1 were also found to be colocalized with
collagen in lymph nodes in vivo. In addition, we were unable to
completely block adhesion of skin-derived DCs to collagen with
an inhibitory anti-␣1 Ab. Thus, it is possible that other receptors
expressed on CD40highCD8␣int pLN-derived DCs, particularly
␣1␤1-negative CD40highCD8␣int pLN-derived DCs, may also mediate adhesion to collagen. It is unlikely that either ␣10␤1 or ␣11␤1,
two recently discovered collagen-binding integrins (35, 36), is responsible for mediating adhesion to collagen because Abs to ␣1
and ␤1 reduced adhesion of the CD40highCD8␣int pLN-derived
subset to similar levels. Although other nonintegrin receptors, such
as CD44 and CD26, are also capable of interacting with collagen
(37, 38), both CD44 and CD26 are expressed at comparable levels
on all CD11chigh DC (data not shown).
The extensive colocalization of ␣1 integrin staining with collagen in LNs suggests that the fibroblastic reticular cells also express
␣1 integrin and most likely use this integrin to interact with collagen in the reticular network. The ␣1 integrin staining pattern is in
sharp contrast to that observed with another collagen-binding integrin, ␣2␤1, which is found predominantly with the HEVs in LN.
The rest of the LN is devoid of ␣2 integrin staining, consistent with
the lack of ␣2 integrin staining on all of the CD11chigh DCs examined by flow cytometry.
Expression of the ␣1 and ␣E integrin subunits also distinguishes
myeloid DCs from lymphoid DCs, as ␣1 and ␣E are both expressed
at higher levels on the lymphoid DC subset. Thus, the lymphoid
DC subset is characterized by higher levels of expression of the ␣1,
␣E, and ␣6 integrin subunits when compared with myeloid DCs.
Although lymphoid DCs express ␣1␤1 at levels comparable to
CD40highCD8␣int pLN-derived DCs, they do not adhere well to
collagen in in vitro adhesion assays. It is possible that, like other
FIGURE 8. Differential conjugate formation and Ag uptake by
CD40highCD8␣int peripheral LN-derived DCs expressing the ␣1␤1 and
␣E␤7 integrins. A, Ag-specific transgenic T cells were isolated and purified
from LNs of OT-I B6.PL mice. DCs were isolated from the pLN of B6
mice and were pulsed in the absence of peptide or in the presence of the
cognate peptide (OVA) or a control peptide (SIY). T cells were incubated
with Ag-pulsed DCs, as described in Materials and Methods, and each
sample was stained with anti-CD11c PE, anti-CD40 PE/Cy5, anti-␣E FITC,
and anti-Thy-1.1 APC. DCs were identified by the expression of high levels of CD40 and CD11c, and could be subdivided based on expression of
␣E. Transgenic OT-I T cells were identified by Thy-1.1 expression. The
degree of conjugate formation was determined by the percentage of each
subpopulation of DCs that also exhibited FL-4 (Thy-1.1) fluorescence. The
mean percentage of duplicate samples, each consisting of three pooled
wells ⫾ SD, is shown. Results are representative of at least three independent experiments. ⴱ, p ⬍ 0.07; ⴱⴱ, p ⬍ 0.01; ⴱⴱⴱ, p ⬍ 0.05. B, Four hours
following the s.c. injection of the fluorescent Ag, E␣RFP, draining and
nondraining LN were harvested and stained for ␣1, CD11c FITC, and
CD40 PE/Cy5. DCs were identified by the expression of high levels of
CD11c and CD40. The degree of Ag uptake was determined by FL-2
fluorescence, and ␣1 integrin expression was determined by FL-4
fluorescence.
hemopoietic cells, lymphoid DCs may require an exogenous signal
that enhances integrin-mediated adhesion (39 – 41). Differential use of
␤1 integrins by DC subsets is also observed in the adhesion of DC
subsets to fibronectin. Although all CD11chigh cells express comparable levels of the fibronectin-binding integrins ␣4␤1 and ␣5␤1, we
consistently observed greater adhesion of CD40highCD8␣int pLNderived DCs to fibronectin. This may reflect the lack of ␣5 integrindependent adhesion of CD40int DCs to fibronectin, as an inhibitory
anti-␣5 mAb partially blocked the adhesion of CD40highCD8␣int
pLN-derived DCs to fibronectin, but had a minimal effect on the
adhesion of myeloid and lymphoid DCs. In general, these results
suggest that blood-borne DC subsets express less functionally active
␤1 integrins than CD40highCD8␣int pLN-derived DCs.
We also examined whether ␣1␤1 and ␣E␤7 integrins were expressed on DCs in the skin or on DCs that migrate from the skin
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FIGURE 7. ␣1␤1 and ␣E␤7 integrin expression on CD40highCD8␣int
pLN-derived DCs is acquired in the LN. Each ear of a B6 mouse was
painted with 50 ␮l of a solution containing 3.3 mM Cell Tracker Green
solution in a 50:50 (v/v) mixture of acetone and dibutylthalate, as described
previously (7, 19). Eighteen hours following the application, DCs were
isolated from pLNs, as described in Materials and Methods. DCs were
stained with anti-CD11c PE, anti-CD40 PE/Cy5, and Abs against either ␣1
(top panel) or ␣E (bottom panel). The DCs stained with Cell Tracker were
identified based on their fluorescence in the FL-1 channel, and represented
27% of the cells in the top panel and 33% of the cells in the bottom panel.
Results are representative of at least three independent experiments.
290
latory cytokines (10). Concomitant with these changes in integrin
expression is a reduction in the ability to take up soluble Ag and
enhanced ability to interact with T cells independent of Ag. It is
interesting to note that the decreased capacity of ␣1␤1⫹␣E␤7⫹
CD11chighCD40high pLN-derived DCs to acquire Ag in vivo was
not associated with decreased ability to form Ag-dependent conjugates with T cells. In fact, ␣1␤1⫹ skin-derived DCs formed
Ag-dependent conjugates at a level comparable to or greater than
skin-derived DCs lacking expression of ␣1␤1 integrin. The identification of two unique subsets of CD11chighCD40high pLNderived DCs is consistent with recent evidence that both immature
and mature skin-derived DCs are present in the LN (49) and
supports a model in which ␣1 and ␣E integrins are markers of a
functionally distinct and, possibly, mature subset of
CD11chighCD40high skin-derived DCs. Acquisition and presentation of soluble Ag by resident skin-derived DCs have recently been
shown to be critical to the initial activation of T cells (12). The
novel DC subset identified in this report may be central to this
process, because these cells express the appropriate integrin receptors that facilitate interaction with collagen, the primary ECM
component found in the reticular network in LNs, and can interact
very efficiently with T cells despite a reduction in the ability to
acquire soluble Ag. Alternatively, the increased Ag-independent
adhesion of this novel DC subset to T cells may be indicative of a
function for this DC subpopulation in maintenance of tolerance in
the periphery.
Acknowledgments
We thank Drs. K. Hogquist, M. Jenkins, D. Mayerova, and J. McCarthy for
reagents as well as helpful comments and suggestions.
References
1. Shortman, K., and Y. J. Liu. 2002. Mouse and human dendritic cell subtypes. Nat.
Rev. Immunol. 2:151.
2. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of
immunity. Nature 392:245.
3. Shimizu, Y., D. M. Rose, and M. H. Ginsberg. 1999. Integrins and the immune
response. Adv. Immunol. 72:325.
4. Metlay, J. P., M. D. Witmer-Pack, R. Agger, M. T. Crowley, D. Lawless, and
R. M. Steinman. 1990. The distinct leukocyte integrins of mouse spleen dendritic
cells as identified with new hamster monoclonal antibodies. J. Exp. Med.
171:1753.
5. Henri, S., D. Vremec, A. Kamath, J. Waithman, S. Williams, C. Benoist,
K. Burnham, S. Saeland, E. Handman, and K. Shortman. 2001. The dendritic cell
populations of mouse lymph nodes. J. Immunol. 167:741.
6. Salomon, B., J. L. Cohen, C. Masurier, and D. Klatzmann. 1998. Three populations of mouse lymph node dendritic cells with different origins and dynamics.
J. Immunol. 160:708.
7. Ruedl, C., P. Koebel, M. Bachmann, M. Hess, and K. Karjalainen. 2000. Anatomical origin of dendritic cells determines their life span in peripheral lymph
nodes. J. Immunol. 165:4910.
8. Puig-Kroger, A., F. Sanz-Rodriguez, N. Longo, P. Sanchez-Mateos, L. Botella,
J. Teixido, C. Bernabeu, and A. L. Corbi. 2000. Maturation-dependent expression
and function of the CD49d integrin on monocyte-derived human dendritic cells.
J. Immunol. 165:4338.
9. Kaldjian, E. P., J. E. Gretz, A. O. Anderson, Y. Shi, and S. Shaw. 2001. Spatial
and molecular organization of lymph node T cell cortex: a labyrinthine cavity
bounded by an epithelium-like monolayer of fibroblastic reticular cells anchored
to basement membrane-like extracellular matrix. Int. Immunol. 13:1243.
10. Gretz, J. E., C. C. Norbury, A. O. Anderson, A. E. Proudfoot, and S. Shaw. 2000.
Lymph-borne chemokines and other low molecular weight molecules reach high
endothelial venules via specialized conduits while a functional barrier limits access to the lymphocyte microenvironments in lymph node cortex. J. Exp. Med.
192:1425.
11. Hayakawa, M., M. Kobayashi, and T. Hoshino. 1988. Direct contact between
reticular fibers and migratory cells in the paracortex of mouse lymph nodes: a
morphological and quantitative study. Arch. Histol. Cytol. 51:233.
12. Itano, A. A., S. J. McSorley, R. L. Reinhardt, B. D. Ehst, E. Ingulli,
A. Y. Rudensky, and M. K. Jenkins. 2003. Distinct dendritic cell populations
sequentially present a subcutaneous antigen to CD4 T cells and stimulate different
aspects of cell-mediated immunity. Immunity 19:47.
13. Ingulli, E., D. R. Ulman, M. M. Lucido, and M. K. Jenkins. 2002. In situ analysis
reveals physical interactions between CD11b⫹ dendritic cells and antigen-specific CD4 T cells after subcutaneous injection of antigen. J. Immunol. 169:2247.
14. Den Haan, J. M., S. M. Lehar, and M. J. Bevan. 2000. CD8⫹ but not CD8⫺
dendritic cells cross-prime cytotoxic T cells in vivo. J. Exp. Med. 192:1685.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
into the LN, because CD11chighCD40highCD8␣int DCs found in
pLN have a phenotype consistent with Langerhans cells (7). However, we did not detect expression of either integrin on Langerhans
cells in skin or on skin-derived DCs that migrate into the LN.
These results are supported by the observation that human epidermal Langerhans cells do not adhere to collagen (42, 43) and suggest that ␣1␤1 and ␣E␤7 integrins, unlike the ␣6␤1 integrin (19),
are not involved in the migration of Langerhans cells from the skin
into LNs. Thus, our results suggest that a subpopulation of
CD11chighCD40highCD8␣int DCs acquires expression of ␣1␤1 and
␣E␤7 subsequent to their migration from the skin into LN. Alternatively, the detection of this CD40high DC subset in pLN may be
due to factors in the spleen and mesenteric LN that reduce CD40
expression or factors in pLN that enhance CD40 expression on
DCs expressing ␣1␤1 and ␣E␤7. However, we believe that this is
unlikely, given that we were not able to identify a nonlymphoid
DC subset that expresses ␣1␤1 and ␣E␤7 in either the spleen or
mesenteric LN. Although the nature of the signals that regulate
integrin expression on CD40highCD8␣int pLN-derived DCs remains undefined, our results clearly suggest that the local LN microenvironment plays a role in DC differentiation and function.
We identified two primary functional differences between
CD11chighCD40high pLN-derived DCs defined by differential expression of ␣1␤1 and ␣E␤7. The first is the increased Ag-independent adhesion of ␣E-positive CD40high DCs to T cells in vitro
when compared with ␣E-negative CD40high DCs. The ability of
DC subsets to form conjugates has not been extensively examined.
Pulsing of CD40high DCs with relevant peptide Ag dramatically
enhanced conjugate formation of both subsets of skin-derived DCs
with OT-I T cells, consistent with the ability of TCR triggering to
enhance integrin-dependent interactions between T cells and APCs
(39, 44, 45). The enhanced ability of ␣E-positive CD40high DCs to
interact with T cells, even in the absence of presentation of a relevant cognate Ag, may be important to our understanding of the
dramatic fluctuations in the motility of naive T cells in LN, as
determined in recent reports using two-photon microscopy (46,
47). The increased Ag-independent adhesion of ␣E-positive
CD40high DCs to T cells, coupled with the presence of this DC
subset in normal pLNs, also suggests the possibility that this DC
subset may be particularly important in the role of DCs in maintaining tolerance to self Ags in the periphery (48). The second
functional difference between these subsets of CD40highCD8␣int
pLN-derived DCs is the ability to take up soluble Ag in vivo.
There was considerably greater Ag uptake in the ␣1-negative DCs
compared with the ␣1-positive CD40highCD8␣int pLN-derived
DCs. This suggests that ␣1-negative CD40highCD8␣int pLN-derived DCs are potent scavengers of soluble Ag and retain the endocytic capacity typically characteristic of peripheral DCs. This
result is consistent with our finding that recent DC migrants from
the skin also lacked expression of ␣1 integrin.
Together, these data suggest a model in which ␣1 and ␣E integrins
define two functionally distinct subsets of CD11chighCD40high DCs in
pLN. We propose that under homeostatic conditions, DCs resident in
the skin lack expression of ␣1 and ␣E and migrate at a low, basal
level to the LN in a manner independent of the expression of these
integrins. After migration into the LN, ␣1 and ␣E integrin expression is induced on a subset of these skin-derived DCs, resulting in
enhanced adhesion of these DCs to the collagen-rich fibers that are
a major structural component of the conduit network. Thus, this
DC subset is uniquely situated in the LN to acquire soluble Ag
carried from tissue to the draining LN through these conduits.
Tight apposition of DCs to these conduits is most likely critical to
effective Ag uptake, as the conduit network provides a significant
physical barrier to the diffusion of soluble Ags and immunoregu-
DCs AND ECM-BINDING INTEGRINS
The Journal of Immunology
33. Ignatius, M. J., T. H. Large, M. Houde, J. W. Tawil, A. Barton, F. Esch,
S. Carbonetto, and L. F. Reichardt. 1990. Molecular cloning of the rat integrin
␣1-subunit: a receptor for laminin and collagen. J. Cell Biol. 111:709.
34. Brand, U., I. Bellinghausen, A. H. Enk, H. Jonuleit, D. Becker, J. Knop, and
J. Saloga. 1999. Allergen-specific immune deviation from a TH2 to a TH1 response induced by dendritic cells and collagen type I. J. Allergy Clin. Immunol.
104:1052.
35. Camper, L., U. Hellman, and E. Lundgren-Åkerlund. 1998. Isolation, cloning,
and sequence analysis of the integrin subunit ␣10, a ␤1-associated collagen binding integrin expressed on chondrocytes. J. Biol. Chem. 273:20383.
36. Velling, T., M. Kusche-Gullberg, T. Sejersen, and D. Gullberg. 1999. cDNA
cloning and chromosomal localization of human ␣11 integrin: a collagen-binding,
I domain-containing, ␤1-associated integrin ␣-chain present in muscle tissues.
J. Biol. Chem. 274:25735.
37. Knutson, J. R., J. Iida, G. B. Fields, and J. B. McCarthy. 1996. CD44/chondroitin
sulfate proteoglycan and ␣2␤1 integrin mediate human melanoma cell migration
on type IV collagen and invasion of basement membranes. Mol. Biol. Cell 7:383.
38. Dang, N. H., Y. Torimoto, S. F. Schlossman, and C. Morimoto. 1990. Human
CD4 helper T cell activation: functional involvement of two distinct collagen
receptors, 1F7 and VLA integrin family. J. Exp. Med. 172:649.
39. Dustin, M. L., and T. A. Springer. 1989. T-cell receptor cross-linking transiently
stimulates adhesiveness through LFA-1. Nature 341:619.
40. Shimizu, Y., G. A. van Seventer, K. J. Horgan, and S. Shaw. 1990. Regulated
expression and binding of three VLA (␤1) integrin receptors on T cells. Nature
345:250.
41. Woods, M. L., and Y. Shimizu. 2001. Signaling networks regulating ␤1 integrinmediated adhesion of T lymphocytes to the extracellular matrix. J. Leukocyte
Biol. 69:874.
42. Le Varlet, B., M. J. Staquet, C. Dezutter-Dambuyant, P. Delorme, and
D. Schmitt. 1992. In vitro adhesion of human epidermal Langerhans cells to
laminin and fibronectin occurs through ␤1 integrin receptors. J. Leukocyte Biol.
51:415.
43. Staquet, M.-J., B. Levarlet, C. Dezutter-Dambuyant, and D. Schmitt. 1992. Human epidermal Langerhans cells express ␤1 integrins that mediate their adhesion
to laminin and fibronectin. J. Invest. Dermatol. 99:12S.
44. Shi, J., T. Cinek, K. E. Truitt, and J. B. Imboden. 1997. Wortmannin, a phosphatidylinositol 3-kinase inhibitor, blocks antigen-mediated, but not CD3 monoclonal antibody-induced, activation of murine CD4⫹ T cells. J. Immunol.
158:4688.
45. Freiberg, B. A., H. Kupfer, W. Maslanik, J. Delli, J. Kappler, D. M. Zaller, and
A. Kupfer. 2002. Staging and resetting T cell activation in SMACs. Nat. Immun.
3:911.
46. Miller, M. J., S. H. Wei, I. Parker, and M. D. Cahalan. 2002. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science
296:1869.
47. Miller, M. J., S. H. Wei, M. D. Cahalan, and I. Parker. 2003. Autonomous T cell
trafficking examined in vivo with intravital two-photon microscopy. Proc. Natl.
Acad. Sci. USA 100:2604.
48. Moser, M. 2003. Dendritic cells in immunity and tolerance: do they display
opposite functions? Immunity 19:5.
49. Geissmann, F., M. C. Dieu-Nosjean, C. Dezutter, J. Valladeau, S. Kayal,
M. Leborgne, N. Brousse, S. Saeland, and J. Davoust. 2002. Accumulation of
immature Langerhans cells in human lymph nodes draining chronically inflamed
skin. J. Exp. Med. 196:417.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
15. Pooley, J. L., W. R. Heath, and K. Shortman. 2001. Cutting edge: intravenous
soluble antigen is presented to CD4 T cells by CD8⫺ dendritic cells, but crosspresented to CD8 T cells by CD8⫹ dendritic cells. J. Immunol. 166:5327.
16. Brown, K. A., P. Bedford, M. Macey, D. A. McCarthy, F. LeRoy, A. J. Vora,
A. J. Stagg, D. C. Dumonde, and S. C. Knight. 1997. Human blood dendritic
cells: binding to vascular endothelium and expression of adhesion molecules.
Clin. Exp. Immunol. 107:601.
17. Ma, J., J.-H. Wang, Y.-J. Guo, M.-S. Sy, and M. Bigby. 1994. In vivo treatment
with anti-ICAM-1 and anti-LFA-1 antibodies inhibits contact sensitization-induced migration of epidermal Langerhans cells to regional lymph nodes. Cell.
Immunol. 158:389.
18. Xu, H., H. Guan, G. Zu, D. Bullard, J. Hanson, M. Slater, and C. A. Elmets. 2001.
The role of ICAM-1 molecule in the migration of Langerhans cells in the skin and
regional lymph node. Eur. J. Immunol. 31:3085.
19. Price, A. A., M. Cumberbatch, I. Kimber, and A. Ager. 1997. ␣6 Integrins are
required for Langerhans cell migration from the epidermis. J. Exp. Med.
186:1725.
20. Schneeberger, E. E., Q. Vu, B. W. LeBlanc, and C. M. Doerschuk. 2000. The
accumulation of dendritic cells in the lung is impaired in CD18⫺/⫺ but not in
ICAM-1⫺/⫺ mutant mice. J. Immunol. 164:2472.
21. Hogquist, K. A., S. C. Jameson, W. R. Heath, J. L. Howard, M. J. Bevan, and
F. R. Carbone. 1994. T cell receptor antagonist peptides induce positive selection.
Cell 76:17.
22. Miyake, S., T. Sakurai, K. Okumura, and H. Yagita. 1994. Identification of collagen and laminin receptor integrins on murine T lymphocytes. Eur. J. Immunol.
24:2000.
23. Noto, K., K. Kato, K. Okumura, and H. Yagita. 1995. Identification and functional characterization of mouse CD29 with a mAb. Int. Immunol. 7:835.
24. Kivens, W. J., S. W. Hunt III, J. L. Mobley, T. Zell, C. L. Dell, B. E. Bierer, and
Y. Shimizu. 1998. Identification of a proline-rich sequence in the CD2 cytoplasmic domain critical for regulation of integrin-mediated adhesion and activation of
phosphoinositide 3-kinase. Mol. Cell. Biol. 18:5291.
25. Inaba, K., M. Inaba, M. Deguchi, K. Hagi, R. Yasumizu, S. Ikehara,
S. Muramatsu, and R. M. Steinman. 1993. Granulocytes, macrophages, and dendritic cells arise from a common major histocompatibility complex class II-negative progenitor in mouse bone marrow. Proc. Natl. Acad. Sci. USA 90:3038.
26. Ardavin, C., L. Wu, C. L. Li, and K. Shortman. 1993. Thymic dendritic cells and
T cells develop simultaneously within the thymus from a common precursor
population. Nature 362:761.
27. Manz, M. G., D. Traver, T. Miyamoto, I. L. Weissman, and K. Akashi. 2001.
Dendritic cell potentials of early lymphoid and myeloid progenitors. Blood
97:3333.
28. Traver, D., K. Akashi, M. Manz, M. Merad, T. Miyamoto, E. G. Engleman, and
I. L. Weissman. 2000. Development of CD8␣-positive dendritic cells from a
common myeloid progenitor. Science 290:2152.
29. Wu, L., A. D’Amico, H. Hochrein, M. O’Keeffe, K. Shortman, and K. Lucas.
2001. Development of thymic and splenic dendritic cell populations from different hemopoietic precursors. Blood 98:3376.
30. Vremec, D., and K. Shortman. 1997. Dendritic cell subtypes in mouse lymphoid
organs: cross-correlation of surface markers, changes with incubation, and differences among thymus, spleen, and lymph nodes. J. Immunol. 159:565.
31. Cepek, K. L., S. K. Shaw, C. M. Parker, G. J. Russell, J. S. Morrow, D. L. Rimm,
and M. B. Brenner. 1994. Adhesion between epithelial cells and T lymphocytes
mediated by E-cadherin and the ␣E␤7 integrin. Nature 372:190.
32. Higgins, J. M. G., D. A. Mandlebrot, S. K. Shaw, G. J. Russell, E. A. Murphy,
Y. T. Chen, W. J. Nelson, C. M. Parker, and M. B. Brenner. 1998. Direct and
regulated interaction of integrin ␣E␤7 with E-cadherin. J. Cell Biol. 140:197.
291