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IMMUNOBIOLOGY
Integrin ␣E(CD103)␤7 influences cellular shape and motility in a
ligand-dependent fashion
Stephanie Schlickum,1,2 Helga Sennefelder,1 Mike Friedrich,1 Gregory Harms,1 Martin J. Lohse,1,3 Peter Kilshaw,4 and
Michael P. Schön1,2,5
1Rudolf
Virchow Center, Deutsche Forschungsgemeinschaft (DFG) Research Center for Experimental Biomedicine; 2Department of Dermatology; and 3Institute
of Pharmacology and Toxicology, University of Würzburg, Würzburg, Germany; 4The Babraham Institute, Cambridge, United Kingdom; and 5Department of
Dermatology and Venereology, University of Göttingen, Göttingen, Germany
While the extravasation cascade of lymphocytes is well characterized, data on
their intraepithelial positioning and morphology are scant. However, the latter
process is presumably crucial for many
immune functions. Integrin ␣E(CD103)␤7
has previously been implicated in epithelial retention of some T cells through
binding to E-cadherin. Our current data
suggest that ␣E(CD103)␤7 also determines shape and motility of some lymphocytes. Time-lapse microscopy showed
that wild-type ␣E(CD103)␤7 conferred the
ability to form cell protrusions/filopodia
and to move in an amoeboid fashion on
E-cadherin, an activity that was abrogated by ␣E(CD103)␤7-directed antibodies or cytochalasin D. The ␣E-dependent
motility was further increased (P < .001)
when point-mutated ␣E(CD103) locked in
a constitutively active conformation was
expressed. Moreover, different yellow fluorescent protein–coupled ␣E(CD103) species demonstrated that the number and
length of filopodia extended toward purified E-cadherin, cocultured keratinocytes,
cryostat-cut skin sections, or epidermal
sheets depended on functional ␣E(CD103).
The in vivo relevance of these findings
was demonstrated by wild-type dendritic
epidermal T cells (DETCs), which showed
significantly more dendrites and spanned
larger epidermal areas as compared with
DETCs of ␣E(CD103)-deficient mice
(P < .001). Thus, integrin ␣E(CD103)␤7 is
not only involved in epithelial retention,
but also in shaping and proper intraepithelial morphogenesis of some leukocytes.
(Blood. 2008;112:619-625)
Introduction
Positioning and locomotion of leukocytes within tissues provide
the basis for the molecular crosstalk with other cells and are
prerequisites for a functional immune system. To date, the
recruitment cascade of initial endothelial adhesion, activation,
firm adhesion, transmigration, and subsequent localization into
the connective tissue is one of the best established concepts in
leukocyte biology.1,2 However, many effector functions of
immunocytes are exerted within parenchymatous organs, mostly
epithelia. It is therefore somewhat surprising that we know
relatively little about locomotion and function-determining
morphogenesis of lymphocytes within epithelial tissues, such as
the epidermis of the skin.3
An adhesion receptor that is thought to mediate retention of
lymphocytes within epithelial tissues is integrin ␣E(CD103)␤7.4
First described 2 decades ago as a selective marker for intestinal
intraepithelial lymphocytes,5 ␣E(CD103)␤7 has been implicated in
epithelial T-cell retention through binding to E-cadherin.6,7 Indeed,
␣E(CD103)-deficient mice exhibited a reduced number of mucosal
intraepithelial T cells.8 However, ␣E(CD103)␤7 has later been
found to be also expressed by some lymphocytes within other
epithelia, such as the epidermis of the skin,9 where it presumably
contributes to recruitment of T cells in inflamed human skin10 as
well as dendritic epidermal T cells (DETCs) in murine skin.11
Thymic DETC precursor cells express integrin ␣E(CD103)␤7
before their migration into the periphery, suggesting that
␣E(CD103)␤7 is involved in guiding tissue-specific epidermal
localization of DETCs.12 Expression of ␣E(CD103)␤7 has been
described on some CD4⫹CD25⫹13,14 and CD8⫹15,16 regulatory
T cells (Treg). In several experimental models ␣E(CD103)␤7 is
involved in guiding tissue localization of lymphocyte subsets in
inflammatory conditions and/or allograft rejection.14,17,18 Although ␣E(CD103)␤7 is highly expressed by some lymphocytes
and CD11high/MHC-IIhigh dendritic cells at mucosal and other
epithelial sites, its role in immune regulation remains largely
elusive. Expression of ␣E(CD103)␤7 appears to be associated
with cytotoxic activity of CD8⫹ T cells in graft-versus-host
disease and allogeneic transplantation.19,20 Along this line,
interactions of ␣E(CD103)␤7 with E-cadherin are crucial for
lysis of E-cadherin⫹ tumor cells by cytotoxic T cells in some
cases.21 Moreover, expression of ␣E(CD103)␤7 is associated
with several important cellular activities such as antigen
presentation22 or stimulation of Treg23 and some mucosal CD8⫹
T cells24,25 by ␣E(CD103)␤7⫹ dendritic cells. It is, however,
currently unclear whether and to what extent ␣E(CD103)␤7 itself
contributes directly to such functions.
Overall, a robust body of evidence has accumulated indicating
that ␣E(CD103)␤7 is involved in tissue-specific retention and/or
effector functions of some immune cells. Yet, it is still unclear
whether ␣E(CD103)␤7 is involved in other cellular functions such
as fine-tuning of shape and motility, similar to what has been shown
for some other integrins,26 which are presumably crucial for the
exertion of effector functions within epithelial compartments. We
Submitted January 22, 2008; accepted March 16, 2008. Prepublished online as
Blood First Edition paper, May 20, 2008; DOI 10.1182/blood-2008-01-134833.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 USC section 1734.
The online version of this article contains a data supplement.
© 2008 by The American Society of Hematology
BLOOD, 1 AUGUST 2008 䡠 VOLUME 112, NUMBER 3
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BLOOD, 1 AUGUST 2008 䡠 VOLUME 112, NUMBER 3
SCHLICKUM et al
present here the first experimental data showing that integrin
␣E(CD103)␤7 is indeed involved in sculpting the shape as well as
stimulating the motility of cells on ligand contact. Thus, integrin
␣E(CD103)␤7 also determines the ligand-directed shape and locomotion of cells, a novel function that extends beyond mere
adhesion and retention on the substrate.
Methods
Plasmids
Yellow fluorescent protein was fused C-terminally to 3 different integrin
␣E-constructs. The integrin ␣E stop codon was eliminated by site-directed
mutagenesis (primer: sense 5⬘-GTCTGCTCCAAGATCAGCCCCCTGCTTC; antisense 5⬘-GAAGCAGGGGGCTGATCTTGGAGCAGAC) using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene,
Amsterdam, The Netherlands). The YFP-gene was amplified by polymerase
chain reaction (PCR) using YFP-primers linked with a NotI restriction site
(sense 5⬘-AGGCTGTGAGGCGGCCGCTAATGGTGAGCAAGGGCGAG,
antisense 5⬘-CTCTAGCGTTGCGGCCGCCTACTTGTACAGCTCGTC)
and Platinum Pfx DNA Polymerase (Invitrogen, Karlsruhe, Germany). The
PCR product as well as the 3-point mutated plasmids were digested with
NotI (NEB, Frankfurt/Main, Germany) and ligated with T4 DNA Ligase
(Invitrogen). The following PCR conditions were used: mutagenesis 95°C
1 minute, followed by 18 cycles 95°C 50 seconds, 60°C 50 seconds, 68°C
9 minutes; YFP 94°C 2 minutes, followed by 30 cycle, 94°C 15 seconds,
58°C 30 seconds, 68°C 1 minute. All plasmids were sequenced (SEQLAB,
Göttingen, Germany).
Cell culture and transfections
K562 cells were cultured in RPMI and PAM212 cells were cultured in
DMEM (PAA, Pasching, Germany). The Nucleofector Kit V (Amaxa,
Cologne, Germany) was used for transient transfections. The transfection
efficiency was determined by flow cytometry. For coating, slides were
overlayered with 1 ␮g/mL recombinant murine E-cadherin (R&D Systems,
Wiesbaden, Germany) in 2 mM CaCl2/PBS over night at 4°C. Kollagenreagens Horm (NYCOMED, Linz, Austria) was diluted 1:1 in PBS and coated
on cover slips overnight at 37°C. For antibody or cytochalasin D treatment
the transfected K562 were incubated for 60 minutes with the 2E7 mAb
(20 ␮g/mL) or cytochalasin D (0.5 ␮M), respectively. As determined by
both trypan blue exclusion and flow cytometry assessing annexin V/propidium iodide staining, cytochalasin D treatment did not affect cell viability
under the conditions used in our study (⬎ 95% viable cells).
formed: mock-transfected cells, 7 experiments; wild-type ␣E(CD103)/␤7,
3 experiments; ␣E-open/␤7, 6 experiments; and ␣E-closed/␤7, 4 experiments.
Mice
The isolation of animal material was approved by governmental
authorities (Regierung von Unterfranken [District Government of
Lower Franconia], Germany) and was performed according to institutional guidelines. Mice (C57/BL6 background) were maintained under
specific pathogen-free conditions.
Epidermal cell suspensions and flow cytometry
Mouse ears were incubated with dispase (Boehringer, Mannheim, Germany) at 6 mg/mL for 1 hour at 37°C. The epidermis was then peeled off
and digested again for 5 minutes at 37°C under gentle stirring in dispase
(6 mg/mL) and DNase (0.1%). The resulting single-cell suspension was
subjected to 2-color flow cytometry using monoclonal antibodies directed
against CD3⑀ (PE-conjugated), CD11a, CD18, CD25, CD29, CD44,
CD49b, CD49d, CD49e, CD49f, and CD103 (FITC-conjugated; BD
Pharmingen, Heidelberg, Germany). Dead cells were excluded by propidiumiodide staining. Cells were analyzed in a FACScan using CellQuest
software (Becton Dickinson, Heidelberg, Germany).
Isolation of epidermal sheets and immunohistochemistry
Ears were removed and depilated. The split skin was floated for 20 minutes
on 0.5 M NH4SCN and the epidermal sheet was peeled off. After fixation in
acetone for 20 minutes at ⫺20°C and washing in phosphate-buffered saline
(PBS), the epidermal sheets were soaked for 90 minutes with antimurine
CD3⑀ (Becton Dickinson) followed by anti-hamster fluorescein isothiocyanate FITC (Vector Laboratories, Burlingame, CA) for 60 minutes. The
sheets were analyzed using an Axioscop 2 microscope equipped with a
Plan-Neofluar 40⫻/0.75 lens (Zeiss), and images were recorded using an
AxioCam HRc (Zeiss). Morphometric analysis was performed using
AxioVision AC software (Zeiss).
Statistical analysis
Data are displayed as mean (⫾ SD or ⫾ SEM as indicated), P values were
determined using the 2-tailed t test, and P values less than .05 (confidence
interval [CI] of 95%) were considered statistically significant. All statistical
analyses were 2-sided.
Confocal microscopy
Cells were cultured for 90 minutes on E-cadherin–coated cover slips.
Imaging was performed by serial 1.0 ␮m Z-steps using a TCS-4D
microscope and SCANware software (Leica, Wetzlar, Germany). For
coculture experiments, 1.5 ⫻ 105 K562 and 4 ⫻ 105 PAM212 were coincubated in RPMI. In other experiments, cryostat-cut sections or epidermal
sheets were overlayered with transfected cells. Confocal microscopy was
performed after 24 hours by serial 0.2 to 0.5 ␮m Z-steps using a DMI6000
microscope equipped with a TCS Sp5 scanner and LAS AF software (Leica
Microsystems, Mannheim, Germany). Morphometric analyses were performed using ImageJ software (version 1.38; National Institutes of Health,
Bethesda, MD).
Time-lapse microscopy
Cells were allowed to attach to E-cadherin for 30 minutes, and were then
monitored for 60 to 90 minutes using an Axiovert 200M microscope
equipped with a LD Achroplan 20⫻/0.4 lens (Zeiss, Göttingen, Germany).
Images were taken every 150 seconds using a CoolSnap ES camera
(Photometrics, Tucson, AZ). Morphometric analyses were performed using
MetaMorph Software (version 6.3r2; Visitron Systems, Puchheim, Germany). The following numbers of independent experiments were per-
Results
Integrin ␣E(CD103)␤7 increases cell motility on E-cadherin
Given that little is known about the role of ␣E(CD103)␤7 for the
motility of leukocytes, we addressed this issue by transfecting cells
with the murine receptor, in which the ␣E(CD103) chain was
locked in different functional conformations by point mutations.27
Unfortunately, transfection of several populations of primary
murine lymphocytes under various conditions did not result in the
expression of functional heterodimers, whereas control proteins
were readily expressed (data not shown). In addition, Jurkat cells
showed constitutive expression of the human ␤7 integrin subunit,
thus precluding functional studies of the murine heterodimer (data
not shown). However, when the erythroleukemia cell line K562
was double-transfected with the wild-type ␣E(CD103) and ␤7
integrin subunits, surface expression and binding to E-cadherin
were readily detected. The overall migration (determined by
single-cell-tracking) on immobilized murine E-cadherin was similar in all transfectant lines expressing ␣E(CD103)␤7 (mean
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BLOOD, 1 AUGUST 2008 䡠 VOLUME 112, NUMBER 3
CELL SHAPE AND MOTILITY AND INTEGRIN ␣E(CD103)〉7
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␣E-WT/␤7 (Figure 1B) as compared with mock transfectants
(P ⬍ .04; Figure 1B). When the cells were transfected with
integrin ␣E-open/␤7, the number of motile cells within the
cultures was even increased by 85% (⫾ 27, P ⬍ .001 compared
with controls; Figure 1B). In addition, the magnitude of cellular
movement and the size of the protrusions formed by the cells
appeared to be markedly greater in transfectants expressing
functionally active ␣E(CD103)␤7 species as compared with the
controls (visualized in Videos S1,S2). Transfection of integrin
␣E-closed/␤7 did not result in enhanced motility of the transfectants (P ⫽ .5 compared with mock-transfected controls; Figure
1B). Culture of the cells on uncoated plastic surfaces, on
collagen-coated surfaces, in the presence of ␣E(CD103)directed antibodies, or cytochalasin D completely abrogated the
increases in amoeboid movement (Figure 1B). Thus, the observed gains of motility were strictly dependent on interactions
of functional ␣E(CD103)␤7 with E-cadherin and appeared to be
relayed through the actin-based cytoskeleton.
Cellular morphology in vitro determined by integrin
␣E(CD103)␤7 on contact with E-cadherin
Figure 1. Expression of integrin ␣E(CD103)␤7 capacitates K562 cells to move in
an amoeboid fashion on E-cadherin. (A) K562 cells were double-transfected with
the murine ␤7 integrin subunit and either wild-type ␣E(CD103) or ␣E(CD103) locked in
a constitutively active (␣E-open) or inactive (␣E-closed) conformation. Control cells
were transfected with the empty vector (pcDNA). Transfectant cells were seeded on
recombinant murine E-cadherin and images were taken every 2.5 minutes. Shape
changes of a representative cell for each condition are shown. ➙ indicates examples
of filopodia and/or cell protrusions. This panel corresponds to Videos S1,S2. Scale
bar ⫽ 10 ␮m. (B) K562 cells were transfected with integrin ␣E-WT/␤7, integrin
␣E-open/␤7, integrin ␣E-closed/␤7, or the empty vector (pcDNA) as detailed in “Cell
culture and transfections.” The transfectants were monitored for 60 minutes on
immobilized E-cadherin ( ), on uncoated plastic surfaces ( ), or on collagen ( ). In
addition, transfectants plated on E-cadherin were treated with a function-blocking
antibody directed against ␣E(CD103) ( ) or were cultured in the presence of
cytochalasin D ( ). The number of cells moving in an amoeboid fashion and
producing protrusions/filopodia is depicted relative to the control (wild-type
␣E(CD103)␤7-transfected cells adhering to fetal calf serum–coated plastic surfaces).
Values shown represent the means ( ⫾ SEM).
;
95 ␮m/h, ⫾ 18), while mock-transfected cells migrated significantly longer distances (average 159 ␮m/h ⫾ 37, P ⬍ .001 compared with either of the ␣E(CD103)␤7 transfectants). However,
some remarkable differences became apparent regarding the motility of cells transfected with the different ␣E(CD103) species at their
site of attachment: Time-lapse microscopy demonstrated that
expression of wild-type (␣E-WT) or a point-mutated ␣E(CD103)
species locked in a constitutively active conformation (␣E-open)
resulted in a significantly increased ability of the cells to move in an
amoeboid fashion on E-cadherin–coated surfaces and to extend
cellular protrusions and filopodia in a highly dynamic manner.
Integrin ␣E-open/␤7 and ␣E-WT/␤7 transfected K562 cells showed
markedly more pronounced changes in morphology as compared
with a constitutively inactive species (␣E-closed/␤7)or mock
(pcDNA3.1) transfected cells (Figure 1A; Videos S1,S2, available
on the Blood website; see the Supplemental Materials link at the
top of the online article). Quantitative analyses demonstrated that
the number of cells showing amoeboid motility was significantly
increased by 50% (⫾ 19) in cultures transfected with integrin
To visualize the formation of cellular protrusions and filopodia in
relation to ␣E(CD103)␤7 expression, ␣E-WT, constitutively active
(␣E-open) and constitutively inactive (␣E-closed) species, respectively, were fused to YFP and transfected into K562 cells. The cells
were cotransfected with the unlabeled ␤7 subunit. As readily
detectable by fluorescence microscopy and flow cytometry, all of
the transfectants showed robust expression of the integrin ␣EYFP/␤7 fusion proteins (data not shown). The expected functional
properties were readily exhibited by the transfectants: Using
wild-type (␣E-WT/YFP/␤7) or the point-mutated species locked in
a constitutively active conformation (␣E-open/YFP) resulted in
significantly increased cell adhesion to E-cadherin (P ⬍ .001 in
both cases as compared with mock-transfected cells), while expression of a constitutively inactive species (␣E-closed/YFP) led to
significantly weaker binding (P ⬍ .01 compared with the control;
Figure 2A), indicating the suitability of the YFP fusion proteins for
further functional experiments. To further ascertain that fusion of
YFP did not alter the function of ␣E(CD103)␤7, additional control
experiments were performed in which ␣E(CD103) was fused to
YFP using either 5 amino acid or 21 amino acid spacer sequences,
both yielding essentially the same results in functional adhesion
experiments (data not shown).
Serial stacks of the K562 cells analyzed by confocal microscopy demonstrated that numerous filopodia were extended by the
cells in the contact zone to E-cadherin–coated surfaces. As
demonstrated by ␣E(CD103) specific antibody-mediated abrogation of filopodia formation, this function was strictly dependent on
␣E(CD103)␤7 (Figure 2B). Strikingly, the number and length of the
filopodia clearly depended on the conformational state of the
␣E-YFP fusion protein transfected. Transfection of ␣E-open/YFP
resulted in formation of the largest filopodia, whereas transfection
of ␣E-WT/YFP led to an intermediate size, and cells expressing
integrin ␣E-closed/YFP exhibited the shortest filopodia (Figure
2C). Statistical analyses verified the significant difference (P ⬍ .02
comparing the ratios cell body/filopodia between ␣E-open/YFP/␤7and ␣E-closed/YFP/␤7-transfected K562 cells). Control experiments using YFP-transfected cells on E-cadherin (Figure 2C) or
integrin ␣E(CD103)/YFP/␤7-transfected cells on collagen (not
shown) did not provoke the formation of filopodia, further indicating that the filopodia of the integrin ␣E(CD103)␤7-transfected cells
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SCHLICKUM et al
BLOOD, 1 AUGUST 2008 䡠 VOLUME 112, NUMBER 3
Figure 2. Expression of functional integrin
␣E(CD103)␤7 influences the cellular shape and induces filopodia formation in a ligand-dependent fashion in vitro. (A) Wild-type or mutant ␣E(CD103) subunits
were fused to YFP as outlined in “Plasmids.” K562 cells
were cotransfected with the wild-type ␤7 subunit and
either wild-type ␣E(CD103)/YFP, ␣E-open/YFP, ␣E-closed/
YFP, or YFP alone as indicated. Adhesion experiments
(determined as bound cells/20⫻ field, n ⫽ 3 experiments) performed on E-cadherin– or FCS-coated surfaces demonstrate the functionality of the integrin ␣E-YFPfusion-proteins. (B) K562 cells cotransfected with wildtype ␣E(CD103)/YFP and ␤7 subunits were plated on
E-cadherin in the presence of a control mAb or a mAb
directed against ␣E(CD103). Images were taken by
confocal microscopy in z-stacks (top panels) and phase
contrast microscopy (bottom panels). Arrows indicate
examples of protrusions/filipodia in the E-cadherin contact zone. (C) The indicated transfectants were seeded
on E-cadherin, and the formation of filopodia was monitored by confocal microscopy. The E-cadherin contact
zone of a representative cell for each condition is shown.
Scale bar ⫽ 10 ␮m. (D) The indicated transfectants were
mixed and cocultured with PAM212 keratinocytes. Analysis was performed by confocal microscopy in z-steps of
0.2 to 0.5 ␮m. One section within the contact zone
between the K562 and PAM212 is depicted, in the top
row the fluorescence and in the bottom row the phase
contrast image of the cells. Arrows indicate examples of
protrusions/filopodia extended toward the keratinocytes.
Scale bar ⫽ 10 ␮m.
were dependent on the specific interaction between ␣E(CD103)␤7
and its ligand, E-cadherin.
Confirming the functional properties in an experimental system
approaching the natural situation within the skin, the formation of
filopodia induced by functionally active ␣E(CD103)␤7 was also
observed when K562 transfectants were cocultured with PAM212,
an E-cadherin-expressing murine keratinocyte line.28 In contrast,
K562 cells expressing ␣E-closed/YFP/␤7 showed only few and
short filopodia, and K562 cells transfected with YFP alone did not
show formation of filopodia when cocultured with PAM212
keratinocytes (Figure 2D).
Generation of an artificial dendritic cell-like phenotype of
leukocytes on natural epidermis through expression of
␣E(CD103)␤7
To more closely mimic the in vivo situation where immune cells
interact with epidermal cells organized in a polarized and stratified
epithelium, we performed 2 complementary series of experiments:
First, K562 cells expressing ␣E(CD103)/YFP constructs and the
wild-type ␤7 subunit were seeded on cryostat-cut sections of
murine skin. In the second series of experiments, epidermal sheets
were prepared from mouse ears, and the sheets were placed
upside-down into plastic dishes. The ␣E(CD103)␤7 transfectants
were then plated onto the exposed undersurface of the epidermal
sheets. Indeed, in both types of experiments some of the transfectant cells expressing active ␣E(CD103)␤7, but not the controls,
formed conspicuous filopodia and protrusions extending clearly
toward the epidermal keratinocytes, thus generating a striking
dendritic cell-like morphology on intact epidermis (examples
depicted in Figure 3A,B).
Integrin ␣E(CD103)␤7 affects the in vivo morphology of
dendritic epidermal T cells (DETC) in mice
The relevance of our findings for properties of naturally occurring
lymphocytes in vivo can be deduced from our further experiments
in which we analyzed wild-type and ␣E(CD103)-deficient mice.8
To address the question whether ␣E(CD103)␤7 affected shape and
morphology of natural cells in vivo, we investigated murine
DETCs, a CD3⫹ cell population of the ␥␦ T-cell lineage that
constitutively expresses ␣E(CD103)␤7 under normal conditions.12
As expected,11 the number of DETCs was reduced in ␣E(CD103)deficient mice as compared with the wild-type controls (littermates,
C57/BL6 genetic background). However, there were conspicuous
morphologic differences between DETCs within the epidermis of
wild-type mice and their counterparts in the skin of ␣E(CD103)deficient mice.
When whole mounts of epidermal sheets were stained by a
CD3-directed monoclonal antibody, it became apparent that
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BLOOD, 1 AUGUST 2008 䡠 VOLUME 112, NUMBER 3
Figure 3. Dendritic morphology of ␣E(CD103)␤7-expressing cells contacting
natural murine epidermis in 2 complementary settings. (A) Epidermal sheets
were separated from the dermis and prepared as outlined in “Isolation of
epidermal sheets and immunohistochemistry.” K562 cells transfected with YFP
alone (left panel) or YFP-fused integrin ␣E-constructs and wild-type ␤7 (␣E-open/
YFP and ␤7 shown here in middle and right panels) were seeded on the exposed
undersurface of the epidermis and the formation of dendrites was visualized by
confocal microscopy. Examples of filopodia extending across or between epidermal keratinocytes are indicated by arrows. The middle and the right panels show
2 different cells at a higher (middle) or lower level (right). The inserts depict the
respective phase contrast images. (B) K562 cells transfected with integrin
␣E-open/YFP and ␤7 were seeded onto cryostat-cut sections of murine skin. The
top row shows a situation where the epidermis is separated from the dermis and
2 transfectant cells are located in the resulting cleft, one cell with contact to the
epidermis and one cell without. Arrows point to examples of dendrites directed
toward the epidermis. The asterisk in the top left panel identifies a transfectant cell
that has no contact to the epidermis and shows no formation of filopodia. In the
bottom row, a transfectant cell is located across the dermo-epidermal junction.
Arrows identify some dendrites extending across the epidermis. The dashed lines
indicate the borders of the epidermis (including stratum corneum in the 2 bottom
panels), and the diamond indicates the location of the viable epidermal cell layers.
Scale bar ⫽ 10 ␮m. These images correspond to Videos S3 to S6.
both number and size of dendrites per cell in ␣E(CD103)deficient mice were significantly reduced as compared with
wild-type animals (Figure 4A). Extensive quantitative morphometric analyses revealed that the dendrites of DETCs from
integrin ␣E-deficient mice spanned an average area of 325 ␮m2
(⫾ 64.6), as compared with 495 ␮m2 (⫾ 91.9) in wild-type
littermates, which was larger by 52.3% (P ⬍ .001; Figure 3B).
In addition, the average number of dendrites per DETC was
reduced significantly in ␣E(CD103)-deficient mice (P ⬍ .001).
DETCs isolated from the epidermis of wild-type mice showed a
significantly higher proportion of cells forming protrusions/
filopodia when plated on E-cadherin as compared with DETCs
isolated from ␣E(CD103)-deficient mice (P ⬍ .003). Again, the
formation of such protrusions was abrogated by antibodies
directed against ␣E(CD103), ␤7, or E-cadherin (Figure S1).
Given that flow cytometric analysis of single-cell suspensions of
CELL SHAPE AND MOTILITY AND INTEGRIN ␣E(CD103)〉7
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Figure 4. Dendritic morphology of dendritic epidermal T cells is influenced by
integrin ␣E(CD103) in vivo. (A) Epidermal sheets of wildtype and integrin ␣E(CD103)deficient mice on the C57BL/6 genetic background (littermates) were prepared and
DETCs were visualized using an mAb directed against CD3 as outlined in “Isolation of
epidermal sheets and immunohistochemistry.” The scale bar indicates 20 ␮m.
(B) The surface area spanned by DETC dendrites in wild-type (left bar) and integrin
␣E-deficient littermates (right bar) was analyzed using ImageJ software. The values
shown represent data from 50 DETCs that were analyzed from 3 mice of
each genotype.
murine epidermis showed no compensatory alterations in other
adhesion molecules including the integrin chains ␣L(CD11a),
␤2(CD18), ␤1(CD29), ␣2(CD49b), ␣4(CD49d), ␣5(CD49e) and
␣6(CD49f) on DETCs of ␣E(CD103)-deficient mice (data not
shown), the morphologic differences between DETCs of
␣E(CD103)␤7-deficient mice and DETCs of wild-type mice can
most likely be attributed to the expression status of ␣E(CD103)␤7.
Discussion
Our results indicate that expression of functional ␣E(CD103)␤7
integrin is important for some cells to adapt and sculpt their
shape on encountering E-cadherin, a ligand that is constitutively
expressed within many epithelial tissues, in a fashion that allows
close contact with ligand-bearing structures. This is the first
example of such a function of a lymphocyte adhesion receptor
that acts primarily within parenchymatous tissues, where many
target cells for immune reactions are located and where effector
functions of immigrating immune cells are exerted. In contrast,
the cascade of lymphocyte extravasation and locomotion within
the connective tissue is well established.1,2 It is therefore
conceivable that this novel functional property of ␣E(CD103)␤7
plays a role for the intraepithelial functions of ␣E(CD103)␤7bearing cells in vivo.
First described as a marker for intestinal intraepithelial
lymphocytes,4-7,29 integrin ␣E(CD103)␤7 has been an enigmatic
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BLOOD, 1 AUGUST 2008 䡠 VOLUME 112, NUMBER 3
SCHLICKUM et al
and tantalizing heterodimeric receptor. Although ␣E(CD103)␤7
is expressed at high levels by CD8⫹ mucosal and epidermal
T cells, it is also found on small subsets of T lymphocytes
elsewhere. Approximately 30% of splenic CD4⫹CD25⫹ regulatory T cells express ␣E(CD103)␤7, apparently independent of
T-cell activation.14,30,31 The receptor has been implicated in
guiding tissue localization of lymphocyte subsets under inflammatory conditions10,14,18 and/or allograft rejection.17,19,32,33 Expression of ␣E(CD103)␤7 appears to be associated with cytotoxic activity of CD8⫹ T cells.19,20,34,35 Along this line,
interactions of ␣E(CD103)␤7 with E-cadherin appear to be
crucial for lysis of autologous E-cadherin⫹ tumor cells by
cytotoxic T cells in some cases.21 Expression of ␣E(CD103)␤7 is
associated with several important cellular activities of mucosal
dendritic antigen presenting cells, such as antigen presentation13,22,36 or stimulation of Treg23 and some mucosal CD8⫹
T cells24,25 by ␣E(CD103)␤7⫹ dendritic cells. Overall, a robust
body of circumstantial evidence suggests an association of
␣E(CD103)␤7 expression with important immune functions. It is
nevertheless currently unclear whether and to what extent
␣E(CD103)␤7 itself contributes directly to such cellular functions, so the precise role of ␣E(CD103)␤7 in immune regulation
still remains largely elusive. However, it appears reasonable to
assume that the proper exertion of all of the above-mentioned
immunologic functions attributed to ␣E(CD103)␤7-expressing
cells requires that the interacting immune cells adapt their shape
according to preformed tissue structures that need to come in
close contact to each other. Based on our results it is now
conceivable that ␣E(CD103)␤7 contributes to this process in
some cell types.
One example of a naturally occurring cell type of the T-cell
lineage, DETCs, has been investigated in this study using
␣E(CD103)␤7-deficient mice and their wild-type counterparts.
Supporting the hypothesis that ␣E(CD103)␤7 facilitates the
formation of cellular protrusions and filopodia “squeezing”
between epidermal keratinocytes, DETCs of ␣ E(CD103)deficient mice showed significantly fewer and shorter dendrites
as compared with wild-type DETCs. Forming dendrites and
taking an appropriate shape are presumably important prerequisites for proper contact formation of DETCs with a maximum
number of neighboring keratinocytes and other epidermal cells
and, therefore, may play a role for at least some of the functions
attributed to DETCs including maintenance of epidermal integrity, epithelial defense, wound healing, regression of skin
tumors, or suppression of cutaneous graft-versus-host disease.37-40 It is therefore conceivable that ␣E(CD103)␤7 is
indirectly involved in such functions through facilitating the
proper shape of DETCs within the epidermis. The hypothetical
concern that the observed functional differences between wildtype and ␣E(CD103)␤7-deficient DETCs might result from
compensatory changes in other adhesion molecules appears
unlikely, because no such changes could be detected in several
candidate molecules. New transfection strategies, such as RNA
electroporation into primary lymphocyte populations,41 might
help to further investigate such issues in the future.
How the ␣E(CD103)␤7 integrin influences the cellular shape
and formation of filopodia is not entirely clear yet, but likely
involves signaling interactions with components of the cytoskeleton, as evidenced by the abrogation in the presence of cytochalasin D. Such signaling events have been demonstrated for some
other integrins including ␣IIb␤3 or ␣1␤1.26,42 Likewise, it is possible
that adaptor molecules contribute to the intracellular transmission
of “outside-in” signals generated through the ␣E(CD103)␤7 integrin, as has also been demonstrated for other integrins such as ␣4␤1
or ␣L␤2.43-45 In any case, the morphologic differences in a defined
population of naturally occurring T cells strongly suggest that
␣E(CD103)␤7 influences the cellular shape not just in transfectionbased artificial systems, but also in vivo, where the situation might
be more complex and might involve a plethora of other adhesive
interactions and/or mediators.
Acknowledgments
This work was supported by a Rudolf Virchow Award and a
research grant from the Deutsche Forschungsgemeinschaft
(M.P.S.). G.H., M.J.L., and M.P.S. are members of the Rudolf
Virchow Center.
Authorship
Contribution: S.S. generated and validated YFP-coupled constructs, performed transfections, video microscopy, animal experiments, and most of the functional experiments, and drafted the
manuscript; H.S. generated YFP-coupled constructs and performed
transfections; M.F., G.H., and M.J.L. critically contributed to the
confocal microscopy, set up experimental conditions, contributed
to the images containing photomicrographs, and made significant
intellectual input to the manuscript; P.K. generated point-mutated
␣E-constructs and provided significant intellectual input to the
manuscript; M.P.S. conceived and planned the study, contributed to
the morphometric analyses, coculture studies, and preparation of
epidermal sheets, and wrote the manuscript. All authors critically
read and approved the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Michael P. Schön, Department of Dermatology and Venereology, Georg August University Göttingen, von
Siebold Str 3, 37075 Göttingen, Germany, e-mail: michael.
[email protected].
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2008 112: 619-625
doi:10.1182/blood-2008-01-134833 originally published
online May 20, 2008
Integrin αE(CD103)β7 influences cellular shape and motility in a
ligand-dependent fashion
Stephanie Schlickum, Helga Sennefelder, Mike Friedrich, Gregory Harms, Martin J. Lohse, Peter
Kilshaw and Michael P. Schön
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