By
From the * Division of Cellular Immunology, National Institute for Medical Research, Mill Hill,
London, NW7 1AA, United Kingdom; and Zeneca Central Toxicology Laboratory, Alderley Park,
Macclesfield, Cheshire, SK10 4TJ, United Kingdom
Topical exposure of mice to chemical allergens results in the migration of epidermal Langerhans cells (LCs) from the skin and their accumulation as immunostimulatory dendritic cells
(DCs) in draining lymph nodes. Epidermal cell-derived cytokines have been implicated in the
maturation and migration of LCs, but the adhesion molecules that regulate LC migration have
not been studied. We hypothesized that integrin-mediated interactions with extracellular matrix components of the skin and lymph node may regulate LC/DC migration. We found that
6 integrins and
4 integrins were differentially expressed by epidermal LCs and lymph node
DCs. A majority of LCs (70%) expressed the
6 integrin subunit, whereas DCs did not express
6 integrins. In contrast, the
4 integrin subunit was expressed at high levels on DCs but at
much lower levels on LCs. The anti-
6 integrin antibody, GoH3, which blocks binding to
laminin, completely prevented the spontaneous migration of LCs from skin explants in vitro
and the rapid migration of LCs from mouse ear skin induced after intradermal administration of
TNF-
in vivo. GoH3 also reduced the accumulation of DCs in draining lymph nodes by a
maximum of 70% after topical administration of the chemical allergen oxazolone. LCs remaining in the epidermis in the presence of GoH3 adopted a rounded morphology, rather than the
interdigitating appearance typical of LCs in naive skin, suggesting that the cells had detached from neighboring keratinocytes and withdrawn cellular processes in preparation for migration,
but were unable to leave the epidermis. The anti-
4 integrin antibody PS/2, which blocks
binding to fibronectin, had no effect on LC migration from the epidermis either in vitro or in
vivo, or on the accumulation of DCs in draining lymph nodes after oxazolone application. RGD-containing peptides were also without effect on LC migration from skin explants.
These results identify an important role for 6 integrins in the migration of LC from the epidermis to the draining lymph node by regulating access across the epidermal basement membrane. In contrast,
4 integrins, or other integrin-dependent interactions with fibronectin that
are mediated by the RGD recognition sequence, did not influence LC migration from the epidermis. In addition,
4 integrins did not affect the accumulation of LCs as DCs in draining
lymph nodes.
Mature lymphoid dendritic cells (DCs)1 are derived from
immunologically immature precursors in nonlymphoid tissues. The best studied example of an immature
DC is the epidermal Langerhans cell (LC). LCs play important roles in the induction of sensitization to contact allergens. After topical application of allergen, LCs are induced
to migrate from the skin to the draining lymph node, where they interact with naive T cells migrating in from
the blood. Accompanying this migration is their maturation
from an antigen-uptake and processing phenotype, typical
of immature DCs in nonlymphoid tissues (1, 2), to an antigen-presenting phenotype typical of DCs in lymphoid tissues (3, 4). Maturation and migration of LCs are central
events in the initiation of cutaneous immune responses to
chemical allergens. Epidermal cell-derived cytokines such
as GM-CSF and IL-1 stimulate the maturation of LCs in
vitro (5, 6). Recent studies have also identified a role for
epidermal cell-derived cytokines in regulating LC migration from the skin to draining lymph nodes. Antibodies to
TNF- The migration of other types of leukocytes is regulated
by specific cell adhesion molecules on the cell surface. The
adhesion molecules that regulate LC migration from the
epidermis to the draining lymph nodes are poorly understood. During migration, LCs dissociate from neighboring
keratinocytes, cross the underlying basement membrane
(BM) into the dermis, enter the afferent lymphatics and
subcapsular sinus leading into the draining lymph node, and relocate in the paracortical or T cell area. In so doing, LCs interact with several different BMs as well as with extracellular matrix (ECM) components of the dermis and lymph
nodes. We hypothesized that integrins on the LC surface
may regulate LC migration from the epidermis to draining
lymph nodes.
Integrins are noncovalently linked Mice.
Young adult, 6-8-wk-old BALB/c mice bred in the
Specific Pathogen Free Units at either the National Institute for
Medical Research or Zeneca Pharmaceuticals were used for these
studies.
Antibodies.
The following antiintegrin antibodies were used
for these studies: PS/2 (anti-murine Peptides.
Gly-Arg-Gly-Asp-Ser (GRGDS) and the control
peptide Gly-Arg-Asp-Gly-Ser (GRDGS) were synthesized on a
model 430A peptide synthesizer (Perkin-Elmer Corp., Norwalk,
CT) using FastMocTM chemistry.
Chemicals and Exposure.
The skin-sensitizing chemical oxazolone
(4-ethoxymethylene-2-phenyloxazol-5-one; Sigma Chemical Co.)
was dissolved in a 4:1 mixture of acetone/olive oil. Groups of
mice received 25 µl of either 1 or 0.5% oxazolone on the dorsum
of each ear.
Cytokine Administration.
Recombinant murine TNF- Antibody Treatment.
Mice were injected intraperitoneally with
100 µl of anti- Preparation of Epidermal Cell Suspensions.
Ears from naive mice
were separated into dorsal and ventral halves with forceps. Dorsal
ear halves were incubated in 0.5% trypsin 1:250 (Sigma Chemical
Co.) in HBSS (GIBCO BRL, Bethesda, MD) for 20 min at
37°C. Epidermal sheets were removed with forceps and washed
three times in RPMI 1640 growth medium (Sigma Chemical Co.),
supplemented with 25 mM Hepes, 50 µg/ml streptomycin, 50 U/ml penicillin, 2 mM glutamine (RPMI), and 20% (vol/vol)
heat-inactivated (56°C for 30 min) FCS. Single cell suspensions
of epidermal cells were prepared by mechanical disaggregation of
the sheets through a stainless steel gauze. The cells were washed
twice and resuspended in RPMI containing 10% FCS (RPMI-FCS) for flow cytometry.
Isolation and Enrichment of Lymph Node DCs.
18 h after topical application of oxazolone, DCs were isolated from auricular
lymph nodes as previously described (16). In brief, mice were
killed by CO2 inhalation, lymph nodes were pooled for each experimental group, and a suspension of lymph node cells was prepared by mechanical disaggregation through a stainless steel
gauze. Cells were washed with 10 ml RPMI-FCS, centrifuged
for 5 min at 300 g, and then resuspended in RPMI-FCS at 5 × 106/ml. DCs were enriched by density gradient centrifugation. 2 ml of Metrizamide (Nycomed, Oslo, Norway) at 14.5% in
RPMI-FCS was layered under 8 ml of lymph node cells and centrifuged (600 g) for 15 min at room temperature. Interface cells
(the low buoyant density fraction) were collected, washed once,
and resuspended in RPMI-FCS. DCs were analyzed by flow cytometry or were assessed by direct morphological examination
using phase-contrast microscopy to determine the frequency of
DCs in low buoyant density fractions. DC frequencies are expressed as number of DCs per node.
Flow Cytometric Analysis.
Epidermal LCs and lymph node DCs
were identified and analyzed for the presence of various cell surface markers by dual color immunofluorescent staining. Cells
(105) were resuspended in PBS containing 0.2% BSA and 0.3%
sodium azide (PBA) and incubated on ice with primary antibody
for 30 min. Cells were washed and centrifuged (300 g for 5 min)
twice with 2 ml of PBA, resuspended in PBA containing either
PE-conjugated anti-rat Ig plus 10% normal mouse serum or PE-SA for biotinylated antibodies, and then cells were incubated for
30 min on ice. Cells were washed once with 2 ml of PBA and
then resuspended in PBA containing 10% normal rat serum for 10 min to block residual anti-rat Ig reactivity. After two washes to
remove rat serum, cells were incubated for 30 min with FITC-conjugated M5/114 (to identify LCs or DCs) and washed twice,
and then a minimum of 10,000 cells were analyzed on a FACStar® flow cytometer. Data were analyzed using FACSplot® software developed by John Green (Computing Laboratory, National Institute for Medical Research).
Skin Explant Assay.
Naive mice were killed by CO2 inhalation and the ears were cut at the base with scissors. The ears were
washed twice with PBS and once with 70% ethanol. Under sterile conditions, ears were spread out on a petri dish, allowed to
dry, and then split into dorsal and ventral halves with forceps.
The dorsal halves were floated individually on 2 ml of RPMI-FCS, or on RPMI-FCS containing antibody or peptide in 16-mm-diameter wells of 24-well cluster trays (Costar Corp., Cambridge, MA). The explants were incubated at 37°C in a 5% CO2
incubator. At various times, explants were removed and epidermal sheets were prepared and analyzed for the presence of LCs as
described below. In experiments designed to test the effect of peptide or antibody on LC migration, three skin explants were used
for each treatment. Control explants were also established in triplicate.
Preparation and Analysis of Epidermal Sheets.
Epidermal sheets
were prepared from naive mice, skin explants, or mice previously
exposed to TNF- prevent the early migration of LCs from the epidermis, the accumulation of DCs in draining lymph nodes,
and the development of optimum contact sensitization in
response to chemical allergens. Conversely, intradermal injection of homologous recombinant TNF-
stimulates the
rapid migration of LCs out of the epidermis and the accumulation of DCs in draining nodes (7). Other cytokines
derived from epidermal cells, and in particular IL-1
, may
act in concert with TNF-
to promote LC migration (10).
/
heterodimers
that form a large family of cell surface adhesion receptors
(11). There are 8
and 16
subunits, divided into subfamilies that share
subunits, giving rise to 22 distinct heterodimers. Integrins are important receptors for adhesion to
ECM proteins, although some integrins, such as the
4 and
2 subunit-containing integrins on leukocytes, mediate
cell-cell adhesion. The epidermal BM comprises a complex
mixture of ECM proteins, including laminin, type IV collagen, and proteoglycans (12). BMs surrounding lymphatics have a similar composition and the paracortical region of
lymph nodes is rich in these ECM proteins as well as fibronectin (13). In this study, we have used blocking antibodies and peptides to determine the potential role of integrin-mediated interactions with two major components of
BM and the ECM, laminin and fibronectin, in regulating
LC migration from the epidermis to draining lymph nodes.
We report that
6 integrins regulate the initial stages of LC
migration out of the epidermis across the underlying BM. In contrast, the
4 integrin or Arg-Gly-Asp (RGD) peptide-mediated interactions with fibronectin are not required.
4 integrin subunit, rat
IgG2b) from the American Type Culture Collection (Rockville,
MD); GoH3 (anti-murine
6 integrin subunit, rat IgG2a), purchased as an affinity-purified antibody from Immunotech (Marseille, France) and obtained as hybridoma supernatant from Dr.
A. Sonnenberg (Amsterdam University, Amsterdam, The Netherlands); EA-1 (anti-mouse
6 integrin subunit, rat IgG2a), a gift
from Dr. B. Imhof (Geneva University, Switzerland); and 346-11A (anti-mouse
4 integrin, rat IgG2a), purchased from PharMingen (San Diego, CA). M5/114 (anti-I-Ad and anti-I-Ed, rat
IgG2b; reference 14) and NLDC-145 (anti-DEC-205, rat IgG2a; reference 15) were used to identify LCs and DCs. MAC193 (antiovine placental lactogen, rat IgG2a), from Dr. G. Butcher
(Babraham Institute, Cambridge, UK) and HRPN11/12a (anti-
horseradish peroxidase [HRP], rat IgG2b) from Dr. S. Hobbs
(Royal Marsden Hospital, London) were used as isotype-matched
control antibodies. Antibodies were purified from tissue culture
supernatants by affinity chromatography using protein G HiTrap
columns (Pharmacia Biotech AB, Uppsala, Sweden). FITC-
(Sigma Chemical Co., St. Louis, MO) and biotin- (Pierce Chemical Co., Rockford, IL) conjugated forms of M5/114 were also
used in some experiments; 50 mg of FITC and 150 mg of biotin
were used per milligram of antibody for conjugation. The following were used as secondary antibodies: PE-conjugated anti-rat Ig
(Southern Biotechnology Associates, Birmingham, AL), FITC-conjugated anti-rat Ig (Sigma Chemical Co.), HRP-conjugated
anti-rat Ig (DAKO Corp., Carpinteria, CA), and streptavidin
(SA) conjugated to either HRP (DAKO Corp.) or PE (Southern
Biotechnology Associates) were used to detect biotinylated reagents.
(specific activity: 2 × 108 U/mg by L929 cytotoxicity assay) was obtained from Genzyme Corp. (Cambridge, MA) as a sterile solution in PBS containing 0.1% BSA as carrier protein. Preparations
were diluted with sterile PBS containing 0.1% BSA and administered using 1-ml syringes with 30-gauge stainless steel needles.
Mice received 30 µl (50 ng) intradermal injections of cytokine
into both ear pinnae. Controls included untreated mice or mice
which had received an equivalent volume of carrier protein
alone.
6 integrin antibody (GoH3, at 100, 200, or 400 µg/ml) or anti-
4 integrin antibody (PS/2 at 200, 400, or 1,000 µg/ml). Animals treated with 100 µg anti-
4 antibody received a
second intraperitoneal injection of 100 µg 8 h after oxazolone
treatment. Control mice were injected intraperitoneally with
equal amounts of isotype-matched control antibody (MAC193 or
HRPN) diluted in sterile PBS. In some experiments both ear pinnae
were injected intradermally with 12 µg of anti-
6 integrin antibody (GoH3) or control antibody (MAC193) in 30 µl of sterile
PBS. In all experiments, one group of animals was left untreated.
by intradermal injection. Dorsal ear halves
were incubated in 0.02 M EDTA in PBS for 1-1.5 h. Epidermal
sheets were fixed in acetone for 20 min at
20°C, washed three
times with PBS, and then incubated for 30 min at room temperature either with anti-MHC class II (M5/114) or biotinylated anti-MHC class II (M5/114) diluted in PBS containing 0.2%
BSA. Sheets were washed three times with PBS and incubated for
30 min at room temperature with either HRP-conjugated rabbit
anti-rat Ig, FITC-conjugated goat anti-rat Ig, or HRP conjugated to SA for biotinylated anti-MHC class II. Sheets were
washed twice with PBS and mounted onto glass slides in Citifluor
(Citifluor Ltd., London, UK) for fluorescence analysis. For immunocytochemical staining, sheets received a further wash with Tris-HCl buffer (50 mM, pH 7.4), and were developed with 1.5 mM
diaminobenzidine (Sigma Chemical Co.) in Tris-HCl buffer for
10 min and washed for a minimum of 10 min with tap water.
The sheets were then mounted onto glass microscope slides, left
to dry for 2 h, dehydrated in alcohol, cleared in Histoclear (National Diagnostics, Atlanta, GA), and mounted in DPX.
We hypothesized that LCs may use distinct integrin adhesion receptors to interact with the underlying BM and other ECM proteins in skin and/or lymph nodes during migration from the skin to draining lymph nodes. We therefore examined epidermal LCs and lymph node DCs for the expression of integrins that mediate adhesion to two of the major components of BM and ECM, laminin and fibronectin (Fig. 1).
LCs were isolated from the ears of naive mice and distinguished from other epidermal cells by MHC class II expression (5, 6). The majority (>70%) of LCs expressed 6
and
4 integrin subunits. The expression of
6 integrins was
determined using two different mAbs, GoH3 and EA-1,
which gave similar results. The staining pattern obtained
with GoH3 was similar using either affinity-purified antibody or hybridoma supernatant. The majority of class II negative epidermal cells, which are primarily keratinocytes, expressed
6 and
4 integrin subunits, as previously reported (17). The expression of
4 integrin subunit was not detectable on isolated LCs, or on other epidermal cells. The expression of NLDC-145 antigen (DEC-205) was also not
detectable on isolated LCs. DCs were isolated from draining (auricular) lymph nodes of oxazolone-sensitized mice
and identified according to size, granularity, expression of
NLDC-145, and by high levels of MHC class II expression (Fig. 1; references 14 and 18). In contrast to LCs, lymph node
DCs expressed the
4 integrin subunit, but did not express
6 or
4 integrin subunits. Expression of the
4 integrin or
NLDC-145 was no longer detectable on DCs after incubation in 0.5% trypsin for 20 min, thus, the lack of
4 integrin and NLDC-145 expression on LCs could reflect loss
of these epitopes during the enzyme digestion used to isolate LCs. In fact, immunocytochemical staining of epidermal sheets showed that LCs in situ express high levels of
NLDC-145 (data not shown). A comparison between LCs
in situ and cytocentrifuged preparations of isolated DCs
showed that LCs expressed much lower levels of the
4 integrin than did isolated lymph node DCs (data not shown).
In summary, the majority of LCs expressed the 6 and
4
integrin subunits whereas DCs did not express either
6 or
4 integrins. Lymph node DCs expressed the
4 integrin
subunit, but much lower levels were found on LCs.
The differential expression of 6 and
4 integrin subunits by LCs and lymphoid
DCs suggested that the
6
4 integrin may regulate the early
stages of LC migration from the epidermis. Conversely, the
higher level of expression of the
4 integrin subunit by
DCs in comparison with LCs raises the possibility that
4
integrins may be involved in DC localization within the
lymph node. To study the roles of these integrins in regulating the initial stages of LC migration from the epidermis,
blocking antibodies to
6 and
4 integrin subunits were
included in the culture medium of skin explants and their
effects on LC migration were determined (Fig. 2). Affinity-purified anti-
6 (GoH3) and anti-
4 (PS/2) integrin antibodies were added to the culture medium at 10, 50, and
100 µg/ml. Control explants were incubated on culture
medium containing isotype-matched control antibody at
100 µg/ml, or on culture medium alone. Explants were incubated for 72 h, the epidermis was removed, and the
number of LCs/mm2 was determined. The number of LCs
in fresh epidermis ranged from 1,000 to 1,200 LCs/mm2.
After 72 h of incubation in culture medium, this number
decreased by ~70% to 300-350 LCs/mm2. Inclusion of
100 µg/ml MAC 193 (rat IgG2a) or HRPN (rat IgG2b) had no effect on the number of LCs remaining in the epidermis after 72 h and these antibodies were therefore used
as isotype-matched controls.
Inclusion of 100 µg/ml anti-6 antibody (GoH3) in the
medium completely prevented the emigration of LCs from
skin explants over a 72-h period. The number of LCs/mm2
remaining in the epidermis (1,193 ± 174) was similar to
that in fresh epidermis (1,168 ± 87). Lower doses of GoH3,
down to 10 µg/ml, also substantially inhibited LC migration. The morphology of LCs remaining in the epidermis
of anti-
6 integrin antibody-treated explants differed significantly from that of LCs in fresh epidermis (Fig. 3). LCs
in
6 integrin antibody-treated skin explants were rounded
in appearance and lacked the interdigitating cellular processes typical of LCs in naive skin. The few LCs remaining in skin explants incubated either in the complete absence of
antibody or in the presence of isotype-matched control antibody showed similar morphologies; LCs were slightly
larger than in naive skin and showed a reduced number of
interdigitating processes. The staining for MHC class II on
LCs in
6 integrin antibody-treated skin and in control
skin explants was more intense than that on LCs in fresh
epidermal sheets.
In contrast to the 6 integrin antibody, the addition of
up to 100 µg/ml antibody to the
4 integrin subunit had
no significant effect on the migration of LCs from skin explants. The number of LCs/mm2 remaining in the epidermis after 72 h of incubation was 254 ± 85, which was not
significantly different from epidermal sheets that had been
incubated either in the complete absence of antibody or in
the presence of the IgG2b control antibody (274 ± 46; Fig. 2). Microscopic examination revealed that the few LCs remaining in
4 integrin antibody-treated explants were morphologically similar to those in control explants incubated
either in the absence of antibody or in the presence of isotype-matched control antibody. As described above, LCs
were larger than in naive skin, showed reduced numbers of
interdigitating processes, and had increased expression of
MHC class II.
The mAb EA-1 recognizes the 6 integrin subunit
and has been shown to inhibit
6 integrin-mediated binding of prothymocytes to thymic blood vessels in mice (19).
However, unlike GoH3, it does not block the interaction
of
6 integrins with laminin, suggesting that
6 integrins
have an alternative ligand to laminin (20). Skin explants
were incubated on culture medium containing
50 µg/ml
EA-1 and the number of LCs was determined after 24 and 72 h (Fig. 4). In contrast to the effects of GoH3, the EA-1
antibody had no effect on the migration of LCs from skin
explants even after 72 h of incubation.
Addition of GRGDS Peptide to Skin Explants Does Not Affect LC Migration.
We were unable to determine the expression of other integrins that may be involved in fibronectin binding, such as the 5 and
1 integrin subunits,
due to lack of available antibodies. Therefore, we have
studied the potential role of the
5
1 integrin in regulating
LC migration using a pentapeptide containing the RGD
sequence which blocks
5
1 integrin-fibronectin interactions (21). Skin explants were incubated on culture medium alone or medium containing either the active pentapeptide GRGDS or the inactive peptide GRDGS (Fig.
4). A maximum dose of peptide (500 µM) was chosen
based on previous studies (22) and this completely inhibited
the binding of EL-4 lymphoma cells to immobilized fibronectin (data not shown). The number of LCs in the epidermis was determined in fresh epidermal sheets and after
24 and 72 h of incubation. Neither GRGDS nor the control peptide had any effect on the migration of LCs from
the epidermis over a 72-h period.
To determine whether 6 integrins play a role in the migration of
LCs from the epidermis in vivo, affinity-purified
6 integrin antibody was administered systemically and its effect
on LC migration was determined after administration of
mouse recombinant TNF-
(Fig. 5). In mice pretreated with
40 µg of isotype-matched control antibody (MAC 193) the
number of LCs/mm2 was reduced from 826 ± 31 in naive
animals to 671 ± 6 after administration of TNF-
, which
is similar to previously reported results (8). However, mice
pretreated with 40 µg anti-
6 integrin antibody GoH3
showed no reduction in the frequency of LCs; the number
of LCs/mm2 at 833 ± 37 was similar to that in naive ear
skin. The morphology of LCs remaining in the epidermis
of
6 integrin antibody-treated mice was different to that
of LCs in naive skin (Fig. 6). A proportion of LCs adopted
a rounded morphology, similar to that of LCs in
6 integrin antibody-treated skin explants, as opposed to the interdigitating appearance typical of LCs. Systemic administration of GoH3 had no effect on the morphology of
resident LCs in untreated skin.
In contrast to the effect of anti-6 integrin antibody, systemic administration of 40 µg anti-
4 integrin antibody
PS/2 had no effect on TNF-
-induced LC migration from
the epidermis (Fig. 5). The morphology of LCs remaining
in the epidermis of anti-
4 integrin antibody-treated mice
was indistinguishable from that of LCs in either naive skin
or skin from mice treated with control antibody (data not
shown).
If 6 integrins are required for LC migration from the
epidermis, in vivo administration of anti-
6 integrin antibody should also inhibit the accumulation of DCs in draining lymph nodes. Anti-
6 integrin antibody was administered either systemically, or directly into the dermis of ear
skin, 2 h before topical application of oxazolone. 18 h after
exposure to oxazolone, draining auricular lymph nodes
were removed and the number of DCs per node was determined. The results from three separate experiments are presented in Fig. 7. In individual experiments, the number of
DCs in untreated, naive mice ranged from 2,000 to 5,000/
node. Topical application of oxazolone consistently increased the number of DCs per lymph node by three- to
fourfold. Administration of the anti-
6 antibody, GoH3,
significantly reduced the number of DCs per lymph node. For example, oxazolone increased the number of DCs per
node from 1,675 to 8,275 in mice treated with control antibody, and intraperitoneal injection of 20 µg of GoH3 reduced DC accumulation to 4,353 per node, representing a
59% inhibition of DC accumulation. 10 µg of GoH3 gave
slightly less inhibition at 37% and the higher dose of 40 µg
did not further inhibit DC accumulation (data not shown). However, 12 µg of antibody administered intradermally was
more effective, inhibiting DC accumulation by 74% (Fig. 7).
Anti-4 antibody had no effect on LC migration from
the epidermis of skin explants, yet this integrin was expressed on lymph node DCs (Fig. 1). The possibility exists
that, rather than being necessary for migration of LCs from
the epidermis,
4 integrins are required for DC migration
into the draining lymph nodes. Therefore, it was of interest
to test the effect of the anti-
4 antibody on lymph node
DC accumulation in vivo.
A single intraperitoneal injection of 20 µg of anti-4 antibody, PS/2, had no effect on oxazolone-induced DC accumulation; the number of DCs per lymph node at 11,322 was similar to that in animals treated with control antibody
(10,355). In the second experiment, a higher dose of antibody was used. A total of 200 µg of anti-
4 antibody was
administered to each animal in two 100 µg doses at 2 h before and 8 h after oxazolone treatment. The numbers of
DCs in the draining lymph nodes 18 h after oxazolone treatment were similar in control and anti-
4 antibody-
treated mice at 8,130 and 7,480, respectively. This lack of
effect was not due to insufficient amounts of functionally
blocking antibody, as lymphocytes taken from these animals were inhibited from binding to immobilized recombinant vascular cell adhesion molecule (VCAM)-1 protein by
70% using standard adhesion assays (reference 23 and data
not shown). Analysis of DCs from anti-
4 antibody-treated mice showed that the cells were saturated with antibody,
such that the staining profile of DCs was similar to that
shown in Fig. 1 (data not shown).
In marked contrast to other types of leukocytes, LCs migrate away from, not towards, a wide range of inflammatory stimuli. After stimulation, LCs dissociate from neighboring keratinocytes, leave their position in the epidermis, pass through the underlying BM into the dermis, enter the afferent lymphatics, and relocate in the T lymphocyte-rich areas of the draining lymph node as mature DCs (24, 25). LCs need to migrate across BMs other than the epidermal BM, including those in the lymphatics and subcapsular sinus that drain into nodes, and we predicted that LCs may use distinct cell adhesion molecules to interact with these BMs during migration from the skin. Therefore, we have studied the expression and function of different integrin receptors for components of the BM on epidermal LCs and on lymph node DCs into which LCs mature.
We identified differential expression by LCs and DCs of
6 and
4 integrin subunits, which dimerize to form a receptor for laminin, and
4 subunit-containing integrins,
which bind fibronectin as well as cell surface molecules such
as VCAM-1 (11). The
6 and
4 integrin subunits were expressed by >70% of LCs, but neither subunit could be detected on lymph node DCs. The
4 integrin subunit was
expressed by all lymph node DCs. It was not possible to
quantitate
4 integrin expression by LCs after isolation, since
the
4 integrin epitope was degraded by trypsin. However,
immunocytochemical analysis of LCs in epidermal sheets
indicated much lower levels of expression than on lymph
node DCs, which agrees with Aiba et al. (26) who reported
low level of
4 integrins on murine LCs. Other authors have
reported the expression of several integrins on LCs, including
4 and
1 integrins (26), but to date, the function of
these integrins on LCs has not been determined. Since
6
and
4 integrins bind to the ECM proteins laminin and fibronectin, respectively (11), we considered that these integrins may regulate the migration of LCs across the epidermal BM into the underlying dermis and/or the subsequent
localization of LC within the draining lymph nodes.
The anti-6 integrin antibody, GoH3, completely inhibited the spontaneous migration of LCs from the epidermis
of skin explants and the rapid migration of LCs from the
epidermis stimulated by TNF-
in vivo. LCs that remained
in the epidermis of
6 integrin antibody-treated skin explants had an altered morphology in comparison with LCs
in naive skin in that the cells were rounded in appearance rather than interdigitating between keratinocytes. LCs in
the epidermis of
6 integrin antibody/TNF-
-treated mice
displayed two distinct morphologies, either a morphology
typical of LCs in unstimulated epidermis, i.e., interdigitating among keratinocytes, or a round morphology. From these
results it can be concluded that
6 integrins are necessary
for the migration of LCs from the epidermis. Since
6 integrins are also expressed on keratinocytes, we cannot formally
exclude an indirect role for
6 integrins on keratinocytes in
regulating LC migration. Upon receiving a stimulus to migrate, LCs may dissociate from surrounding keratinocytes, for example by downregulating E-cadherin expression (29),
and move towards the BM, but be prevented from traversing it by the anti-
6 integrin antibody GoH3, which blocks
binding to laminin. The LCs therefore become trapped in
the epidermis, but nevertheless free from keratinocytes. Ultrastructural analyses will be required to determine the precise location of these rounded LCs with respect to the BM.
The increased expression of MHC class II on
6 integrin- blocked LCs suggests that they may mature in this location.
Preliminary experiments (not shown) indicate that other
markers associated with the maturation of LCs are upregulated on
6 integrin-blocked LCs. The findings in this
study suggest that interactions with laminin in the BM may
not be required for LC maturation.
The mAb GoH3 blocks the interaction of 6
1 and
6
4
integrins with laminin (30). The only defined ligands to date
for
6 subunit-containing integrins are laminin (31), although recent studies with the EA-1 antibody suggest the
existence of an alternative ligand. EA-1 recognizes the
6
subunit of both
6
1 and
6
4 integrins and inhibits
6 integrin-mediated migration of prothymocytes from the bloodstream into the thymus of mice. However, EA-1 does not inhibit
6
1 or
6
4 integrin-dependent binding to laminin (19, 20). In contrast to GoH3, the EA-1 antibody did
not inhibit LC migration from the epidermis, suggesting
that
6 integrin interactions with laminin, as opposed to
other ligands, may regulate this step.
The 6 integrin subunit has two splice variant forms,
6A and
6B, which differ in their cytoplasmic domains
(32). Both
6A and
6B associate with the
1 and
4 integrin subunits to form
6A
1,
6B
1,
6A
4, and
6B
4 integrins (30). All four integrins bind to laminin and differences in the ligand binding specificities of the
6A and
6B
variants have not been detected (30). However, it is of
considerable interest which
subunit is used to form the
6 integrin heterodimer on LCs. The results presented here
demonstrate that >70% of LCs express the
6 integrin and
>70% also express the
4 integrin. Based on this finding, it
can be speculated that the
6
4 integrin is found on >70%
of LCs, particularly as there are currently no other
subunits known to dimerize with the
4 subunit. However,
expression of the
1 integrin subunit on LCs also needs to
be determined. Antibodies to the mouse
1 integrin subunit were not available for such analyses and therefore, we
cannot conclude which
6 integrin is used by LCs for migration. There has been one report demonstrating that all
human epidermal LCs express
1 integrin (28), however,
the same group reported a complete absence of
4 integrin
on these cells (33).
In epithelial tissues, the 6
4 integrin is a key structural
component of hemidesmosomes (34) which mediate stable
interactions between epithelial cells and the underlying BM
and are responsible, in part, for maintaining the functional
integrity of the tissue (35). The
6
4 integrin has also been
implicated in migratory events, including the migration of
keratinocytes during wound healing (36). The apparently
contradictory roles of the
6
4 integrin in the stable adhesion and in the migration of keratinocytes may be explained by the finding that epidermal growth factor receptor signaling in keratinocytes results in phosphorylation of
multiple tyrosine residues on the
4 cytoplasmic tail, which
causes the disruption of hemidesmosomes and
6
4 integrin-mediated migration (37). A change in the activation
status of
6 integrins on LCs may regulate migration in that
the integrin is normally held in an inactive conformation
on resident LCs but after stimulation is converted to an active conformation allowing binding to laminin, thus facilitating migration across the BM. Alternatively,
6 integrins could be expressed in the active form on resident LCs but
this is of no biological relevance as LCs are trapped within
a keratinocyte matrix.
Since we were unable to determine the expression of
other integrin receptors on LCs for fibronectin (such as
5
1), we used RGD peptides to study their potential role
in regulating LC migration. The addition of a synthetic
RGD-containing peptide to the culture medium of skin
explants had no effect on LC migration. RGD is a consensus binding sequence in many integrin ligands, including fibronectin, fibrinogen, vitronectin, von Willebrand factor,
and thrombospondin, but not laminin, and binding of
3
1,
5
1,
v
1,
v
3,
v
5,
v
6, and
IIb
3 integrins to their
ligands is dependent on this sequence (11, 21). The lack of
effect of RGD peptides suggests that the
5
1 integrin, as
well as other RGD-dependent
1 and
3 integrins, does
not regulate LC migration from the epidermis.
Addition of antibodies against the 4 integrin also had no
effect on LC migration either in vitro or in vivo. Although
we could not quantitate
4 integrin expression on LCs, it
was substantially lower than on lymph node DCs. We did
not identify which of the two
4 subunit-containing integrins is expressed by DC (i.e.,
4
1/very late antigen
(VLA)-4, or
4
7) but the PS/2 antibody is an effective inhibitor of both (38, 39) and blocks binding to all ligands
identified, including fibronectin, VCAM-1, mucosal address in cell adhesion molecule (MAdCAM)-1, and the
4
integrin subunit itself (39). The PS/2 antibody is also an
effective inhibitor of
4 integrins on mouse leukocytes in
vivo (42). The lack of effect of this antibody indicates that
4 integrin-mediated interactions with fibronectin or with
any other ligand are not required for the initial stages of LC
migration from the epidermis. However, the skin explant
model used here does not study the subsequent stages of
migration of LCs into the draining lymph nodes, and for
this reason we studied the effect of integrin antibodies on the accumulation of DCs in draining lymph nodes in vivo.
Previous studies have shown that systemic administration
of antibodies to TNF- 2 h before sensitization with oxazolone inhibited the accumulation of DCs in draining
lymph nodes in response to the chemical allergen (9). Therefore, we used a similar model to determine the effects of
anti-
6 integrin and anti-
4 integrin antibodies on DC accumulation. Intraperitoneal administration of 40 µg of
anti-
6 integrin antibody GoH3 maximally inhibited DC
accumulation by 60%. Intradermal injection of 12 µg of
GoH3 directly into the ear gave a slightly greater inhibition
of 74%. The incomplete inhibition may be explained by
the fact that some DCs may come directly from the dermis
and/or the bloodstream. Although lymph node DCs do
not express
6 integrins, it is not possible to conclude that
6 integrin-mediated interactions are not involved in the
migration process once the cells have left the epidermis,
since we do not know precisely when
6 integrin is downregulated during maturation into DCs.
In contrast, saturating doses of anti-4 integrin antibody
had no effect on DC accumulation. It is possible that this
antibody affects the precise localization of DCs within the
lymph node paracortex and further experiments are required to test this hypothesis.
In summary, this study has reported the expression of the
6 and
4 integrin subunits on murine LCs and has demonstrated, for the first time, the direct involvement of
6 integrins in regulating LC migration from the epidermis.
Address correspondence to Ann Ager, Division of Cellular Immunology, National Institute for Medical Research, Mill Hill, London, NW7 1AA, UK. Phone: 44-181-959-3666, ext 2465; FAX: 44-181-913-8529; E-mail: a-ager{at}nimr.mrc.ac.uk
Received for publication 26 June 1997 and in revised form 8 September 1997.
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