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Dermatology Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, and LPS mobilize LC from epidermis and
presumably attenuate LC-keratinocyte adhesion. To determine whether
these mediators modulated LC E-cadherin-dependent adhesion directly, we
characterized their effects on LC-like dendritic cells expanded from
murine fetal skin (FSDDC). FSDDC were propagated from day 16 C57BL/6
fetal skin and isolated as aggregates (FSDDC-A) in which homophilic
adhesion was mediated by E-cadherin. IL-1, TNF-, and LPS induced
dissociation of FSDDC-A that began within 4 to 8 h and was
complete within 20 h. Anti-IL-1RI mAb inhibited disaggregation
caused by IL-1 and IL-1ß, but not that induced by TNF- or LPS.
Anti-TNF- mAb inhibited the effect of TNF- and LPS, but not that
caused by IL-1 or IL-1ß. Flow cytometry of FSDDC-A revealed that
IL-1, TNF-, and LPS induced increased expression of MHC class II,
CD40, and CD86 and decreased E-cadherin expression that was temporally
related to dissociation of aggregates. IL-1 and TNF- caused a rapid
reduction in FSDDC E-cadherin mRNA levels that preceded the decrease in
E-cadherin surface expression. These results demonstrate that cytokines
that induce LC emigration in vivo act directly on LC-like cells in
vitro, reduce E-cadherin mRNA levels, down-regulate E-cadherin surface
expression, and induce a loss of E-cadherin-mediated adhesion. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Much of what is known about DC localization and trafficking comes from
studies of Langerhans cells (LC), the epidermal contingent of the DC
lineage. We previously demonstrated that LC express high levels of
E-cadherin (4, 5) and that E-cadherin mediates selective adhesion of LC
to keratinocytes in vitro (4). We hypothesize that E-cadherin also
plays an important role in LC-keratinocyte adhesion and LC localization
in vivo. The results of several series of experiments are consistent
with this concept. First, among DC, E-cadherin is expressed only by LC
and a subpopulation of skin draining lymph node DC that may be derived
from LC (6). Second, application of contact allergens that mobilize LC
from skin induced a decrease in E-cadherin expression on a substantial
subset (
40%) of activated LC in situ (7). Previous studies
demonstrated that, after exposure to contact allergens, a subpopulation
of LC displays an activated phenotype with characteristic shape
changes, increased MHC class II expression, and enhanced APC activity
(8, 9). E-cadherin levels on activated LC (i.e., LC expressing
increased levels of MHC class II Ag) were only 15 to 20% of those
expressed by LC in normal skin and were similar to levels on cultured
LC, LC that migrated from skin explants (7), and lymph node DC that may
be derived from LC (6). Although these data suggest that
E-cadherin-mediated LC-keratinocyte adhesion may decrease as a
consequence of LC activation and that decreased adhesion may result
from reduced E-cadherin expression, direct evidence in support of this
concept is lacking.
Several lines of investigation implicate IL-1 and TNF- in the
activation and mobilization of LC (9, 10, 11, 12). Application of contact
allergens that induce activation and emigration of LC stimulated
accumulation of mRNA, encoding a number of cytokines (including IL-1
and TNF-) in epidermis (9). In addition, dermal injection of IL-1 or
TNF- induced a decrease of LC in epidermis and an increase in DC in
draining lymph nodes (10, 11, 13, 14). Finally, administration of
anti-IL-1ß and anti-TNF- Ab inhibited contact
allergen-induced sensitization and LC redistribution (9, 15). Recent
studies indicated that IL-1, TNF-, and LPS mobilized nonlymphoid DC
from kidney and heart, as well as epidermis (16). We previously
demonstrated that cutaneous injection of IL-1 and TNF-
down-regulated E-cadherin expression by activated LC in situ (7).
However, these studies did not allow us to determine whether IL-1 or
TNF- acted directly on LC to decrease E-cadherin expression or
indirectly via keratinocytes or other cells.
Studies of LC E-cadherin biology have been hampered by the lack of
availability of large numbers of cells with appropriate
characteristics. We recently described a primary culture system that
allowed the expansion of LC-like DC (FSDDC) from fetal murine skin
(17). FSDDC were propagated from day 16 C57BL/6 fetal skin in medium
containing GM-CSF and CSF-1. Aggregates of E-cadherin+
FSDDC (FSDDC-A) that resembled LC in terms of morphology, phenotype,
and function and that exhibited E-cadherin-dependent homophilic
adhesion were readily isolated (17). Like LC (18), FSDDC-A
spontaneously matured into interdigitating DC-like cells in vitro,
manifested by a characteristic surface phenotype, cytokine profile, and
acquisition of potent allostimulatory activity. This in vitro
maturation, like that of LC (4), was accompanied by decreased
E-cadherin surface expression and by loss of E-cadherin-mediated
adhesion (17). In the present study, we used this in vitro system to
characterize effects of inflammatory mediators that activate and
mobilize LC in vivo on E-cadherin-mediated adhesion in LC-like DC.
Herein we demonstrate that the epidermal proinflammatory cytokines IL-1
and TNF- act on LC-like DC to regulate E-cadherin function by
decreasing steady-state E-cadherin mRNA levels, thereby reducing
E-cadherin surface expression and attenuating E-cadherin-mediated
adhesion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LC-like FSDDC were isolated as aggregates (FSDDC-A) from GM-CSF- and CSF-1-supplemented primary cultures of day 16 C57BL/6 murine fetal skin as previously described (17, 19). When necessary, FSDDC-A were dissociated with trypsin in EDTA (0.01% trypsin in calcium- and magnesium-free HBSS containing 1 mM EDTA for 30 min at 37°C) to allow enumeration of cells. FSDDC aggregates were subcultured in cytokine-supplemented DC medium at 5 x 105 cells/ml in T-25 flasks or 96-well plates as indicated.
Microscopy
FSDDC aggregates were subcultured for 18 h in T-25 flasks
in the presence or absence of murine recombinant IL-1ß (10 ng/ml;
Genzyme, Cambridge, MA), TNF- (10 ng/ml; Genzyme), or LPS (100
ng/ml). (Escherichia coli K235-derived LPS (<0.008%
protein), prepared as described (20), was provided by Dr. Stephanie
Vogel, Uniformed Services University of the Health Services, Bethesda,
MD.). Morphologic changes were documented using an inverted phase
photomicroscope (Axiovert 405 M; Carl Zeiss, Oberkochen, Germany)
equipped with Hoffman modulation contrast optics (Modulation Optics,
Greenvale, NY).
Quantitative disaggregation assay
FSDDC aggregates were seeded into round-bottom 96-well plates at
105 cells/well and incubated for 18 h in the
presence or absence of murine recombinant IL-1, IL-1ß, TNF-,
IL-6, or IL-10 (10 ng/ml each; Genzyme), human recombinant TGF-ß1 (10
ng/ml; R&D Systems, Minneapolis, MN), LPS (100 ng/ml); or cytochalasin
D (CCD; 10 µg/ml; Sigma) in a final volume of 0.2 ml/well. All
cytokines contained less than 5 pg LPS/µg protein (LAL-test,
BioWhittaker, Walkersville, MD). Well contents were subsequently
harvested by limited trypsinization (0.01% trypsin for 15 min at
37°C) in the presence of calcium (which protects cadherins from
trypsin digestion), added to 10 ml PBS, and particles were enumerated
with a Coulter Counter (Coulter Electronics, Hialeah, FL). Counts
obtained in wells without addition of cytokines
(Co) and in wells harvested with trypsin
(0.01%) in 1 mM EDTA (C100, representing
maximal disaggregation) were determined in pentuplicate. Disaggregation
(Cx) induced by cytokines, LPS, or CCD
was determined in triplicate and expressed as a percentage of maximal
disaggregation according to the following equation: X% =
(Cx - C0) x
(C100 -
C0)-1 x 100.
Quantitation of cytokine mRNA and protein
Total RNA was isolated from FSDDC-A after incubation for 18
h in the presence or absence of LPS (100 ng/ml) using RNAzol B
(Tel-Test, Friendswood, TX). Cytokine and control mRNAs were
quantitated using a multiprobe RNase protection assay system
(RiboQuant; PharMingen, San Diego, CA) using the protocol provided by
the manufacturer. Briefly, [32P]UTP-labeled
riboprobes were transcribed from cDNA templates using T7 polymerase
(Promega, Madison, WI) and allowed to hybridize to sample mRNA
overnight at 45°C. After treatment with RNase A and RNase T,
protected fragments were resolved in denaturing 8% polyacrylamide gels
and detected by autoradiography. Two cDNA template sets were used: mCK2
(IL-1, IL-1ß, IL-1RA, IL-6, IL-10, IL-12 (p35), IL-12 (p40),
IFN-
, and MIF) and mCK3 (IL-6, TNF-, TNF-ß, LTß, IFN-ß,
IFN-, TGF-ß1, and TGF-ß2). Five micrograms of total RNA from
FSDDC-A or from yeast (negative control) and 2.5 to 3 x
105 cpm of the radiolabeled probe was used per
lane.
Spontaneous and LPS-induced (or cytokine-induced) cytokine production
was measured in FSDDC supernatants using ELISA kits for murine IL-1,
IL-1ß, and TNF- (Genzyme). FSDDC-A were subcultured in 24-well
plates at 106 cells/ml in the presence or absence of LPS
(100 ng/ml), IL-1, IL-1ß, or TNF- (each at 10 ng/ml), and cell
supernatants were obtained at 24 h. To quantitate cell-associated
IL-1, washed FSDDC were resuspended in PBS containing 2.5 mM EGTA, 2.5
µM p-nitrophenyl-p-guanidinobenzoate (Sigma),
and Complete protease inhibitor mixture (Boehringer Mannheim,
Indianapolis, IN) and subjected to four freeze/thaw cycles (21). Cell
debris was sedimented (15,000 x g for 30 min), and the
soluble fraction was recovered. FSDDC supernatants and lysates were
stored at -70°C until analysis.
Abs and flow cytometry
Anti-IL-1RI mAb (JAMA; 22 , anti-IL-1 mAb (ALF; 23 , and anti-IL-1ß mAb (B122; 24 were provided by Drs.
Robert Schreiber and David Chaplin (Washington University School of
Medicine, St. Louis, MO), and anti-TNF- mAb (hamster IgG) was
purchased from Genzyme. The neutralizing capacity of each mAb was
determined using recombinant cytokines. LPS contamination was <5
pg/100 µg protein (LAL-test; BioWhittaker). Hybridomas producing
anti-I-Ab (Y3P, mouse IgG2a) and
anti-I-Ad (MKD6, mouse IgG2a) were obtained from the
American Type Culture Collection (Rockville, MD). Anti-CD16/CD32
(2.4G2, rat IgG2b) was provided by Julie Titus (National Cancer
Institute, Bethesda, MD), and anti-E-cadherin (ECCD-1, rat IgG1;
ECCD-2, rat IgG2a) by Dr. Masatoshi Takeichi (Kyoto University, Kyoto,
Japan). Anti-CD40 (HM40-3, hamster IgM), anti-CD45 (30F11.1, rat
IgG2b), anti-CD86 (GL-1, rat IgG2a), and isotype controls were
purchased from PharMingen as purified biotin- or FITC-modified mAb.
Phycoerythrin-streptavidin was obtained from Tago (Burlingame, CA). Rat
mAb were purified from ascites or hybridoma supernatants using
immobilized protein G (Pierce, Rockford, IL). Mouse mAb were purified
from supernatants using immobilized protein A (Pierce). Y3P and MKD6
were conjugated with FITC (Sigma) as described (25).
FSDDC-A were subcultured for 18 h in round-bottom 96-well plates in the presence or absence of LPS or cytokines. Before staining, FSDDC were dissociated in HBSS containing 1 mM EDTA (30 min at 37°C) in 10% chelated FBS (26). For multicolor flow cytometry, cells were suspended in cold PBS containing 5% FBS and 0.02% NaN3, preincubated with saturating concentrations of 2.4G2, and then serially incubated with saturating concentrations of FITC-mAb, Bio-mAb, and phycoerythrin-streptavidin. For single-color flow cytometry, cells were incubated with hybridoma supernatants and FITC-modified, affinity-purified (Fab')2 fragments of goat anti-rat IgG (Tago). Stained cells were analyzed using a FACScan flow cytometer equipped with Cellquest software (Becton Dickinson, Mountain View, CA). Propidium iodide-permeable (nonviable) cells were excluded from the analysis.
Northern blot analysis
Total RNA was isolated from FSDDC using RNAzol B, denatured,
fractionated by formaldehyde/agarose gel electrophoresis (10
µg/lane), and transferred to nylon membranes (Hybond; Amersham,
Arlington Heights, IL). Hybridization was performed overnight at 42°C
with random-primed [32P]dCTP-labeled murine
E-cadherin cDNA (Ref. 27; supplied by Dr. John Stanley, University of
Pennsylvania, Philadelphia, PA) in 50% formamide, 10% dextran
sulfate, 1% SDS, and 100 µg/ml salmon sperm DNA. Blots were
subsequently stripped and probed with [32P]dCTP-labeled
rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA (provided by
Dr. Koichi Suzuki, National Institute of Diabetes, Digestive and Kidney
Diseases, National Institutes of Health, Bethesda, MD). Relative
amounts of E-cadherin and GAPDH mRNA were estimated using a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). For dose-response
studies, FSDDC-A were incubated for 18 h with various
concentrations of IL-1ß or TNF- before preparation of RNA. For
time course studies, cytokines (10 ng/ml) were added at various times,
and all cells were harvested simultaneously by vigorous trituration. An
aliquot of each sample was prepared for flow cytometry as described
above.
Statistical analysis
The statistical significance of differences in the disaggregation assay was calculated using the paired Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Intact aggregates of FSDDC (FSDDC-A) were harvested from primary
cultures of day 16 C57BL/6 fetal skin cells maintained for 2 wk in
GM-CSF- and CSF-1-supplemented medium by limited trypsinization in
calcium-containing medium and separated from single cells by 1 g
sedimentation. Within 3 h after initiating subcultures, FSDDC-A
adhered loosely to tissue culture flasks, and cells at the periphery of
the aggregates began to exhibit sheetlike processes and dendrites.
After 18 h, small numbers of cells with pronounced dendritic
morphology were released from the aggregates. However, most cells
remained tightly clustered (Fig. 1
A). We have previously
shown that intercellular adhesion in these aggregates is mediated by
the homophilic adhesion molecule E-cadherin and that cells in FSDDC
aggregates can adhere to other cells via an E-cadherin-dependent
mechanism (17).
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) and LPS have been
implicated in LC activation and in the mobilization of LC (and other
DC) from nonlymphoid tissues (7, 9, 10, 11, 12, 13, 14, 16), we investigated the
effects of these agents on E-cadherin-mediated adhesion in FSDDC-A.
Treatment of FSDDC-A with IL-1ß (Fig. 1
B), TNF-
(Fig. 1C), or LPS (Fig. 1D) for
18 h resulted in almost complete dissociation of FSDDC
aggregates.
The ability of IL-1, TNF-, and LPS to modulate E-cadherin-mediated
adhesion in FSDDC-A was confirmed using a Coulter counter-based
disaggregation assay (Fig. 2A). In addition, we
determined that the effects of these epidermal proinflammatory
cytokines were selective in that other cytokines (including IL-6,
IL-10, and TGF-ß1) did not induce significant disaggregation. As
expected, CCD, an inhibitor of actin polymerization (28), also induced
complete dissociation of FSDDC-A. This is consistent with earlier
studies that demonstrated that classical cadherins mediate adhesion
only if they are physically linked to a functional actin-myosin
cytoskeleton (29). Dose-response studies revealed that half-maximal
disaggregation of FSDDC was induced by 0.2 ng/ml IL-ß and 1 ng/ml
IL-1, TNF-, and LPS, with maximal disaggregation occurring at 10
to 100 ng/ml (Fig. 2B). Time course studies
demonstrated that cytokine- and LPS-induced disaggregation began after
a lag period of 4 h, was half maximal at 12 to 16 h, and was
complete by 20 h (Fig. 2C). In contrast, CCD
induced almost complete dissociation within 4 h (Fig. 2C).
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LPS induces several cytokines in cells of the DC lineage (30, 31, 32, 33).
To characterize the cytokine profile of FSDDC-A, we utilized a
multiprobe RNase protection assay that allowed relative quantitation of
14 different cytokine mRNAs (Fig. 3). The
predominant cytokine mRNAs in FSDDC-A subcultured for 18 h in DC
medium alone were those encoding TNF-, TGF-ß1, and MIF. Weak
signals for IL-1, IL-1ß, IL-1RA, and (at longer exposures) IL-12
mRNAs were also detected. In comparison, FSDDC incubated for 18 h
in the presence of LPS (100 ng/ml) contained dramatically increased
amounts of IL-1, IL-1ß, and IL-1RA mRNA. MIF, TNF-, TGF-ß1,
and IL-12 (data not shown) mRNA levels were comparable in LPS-treated
and control cells.
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, IL-1ß, and TNF- were quantitated in 24-h
FSDDC-A supernatants (and IL-1 in cell lysates) by ELISA. FSDDC-A
released small amounts of TNF- into culture supernatants in the
absence of LPS and considerably more after LPS treatment (Table I). In contrast, little if any IL-1 or
IL-1ß was detected in FSDDC-A supernatants, even after LPS treatment.
Significant amounts of cell-associated IL-1 and IL-1ß were present
in FSDDC-A after stimulation with LPS, however. Interestingly, IL-1
and IL-1ß did not induce TNF- production by FSDDC-A, and TNF-
did not cause significant amounts of IL-1 to be released into the
medium.
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To characterize the involvement of IL-1 and TNF- in the
dissociation of FSDDC-A induced by IL-1, TNF-, and LPS, we assessed
the ability of mAb directed against IL-1, IL-1ß, IL-1RI, and
TNF- to block cytokine- and LPS-induced FSDDC-A disaggregation.
Cytokine and Ab concentrations were titrated such that complete
inhibition of the action of the relevant cytokine was obtained.
Anti-IL-1ß, anti-IL-1RI, and anti-TNF- were used at 50
µg/ml, and anti-IL-1 was used at 0.3 µg/ml. Anti-cytokine or
anti-IL1RI mAb alone had no activity in this assay at the
concentrations tested. As shown in Fig. 4, anti-IL-1RI mAb inhibited the
disaggregation caused by IL-1 (3 ng/ml) and IL-1ß (3 ng/ml) but
not that induced by TNF- (3 ng/ml) or LPS (3 ng/ml). Anti-IL-1
and anti-IL-1ß mAb inhibited the activity of IL-1 and IL-1ß,
respectively, but had no effect on the activity of TNF- or LPS.
Anti-TNF- mAb inhibited the effect of TNF- and LPS but not that
due to IL-1 or IL-1ß. Thus, TNF- and IL-1 induced dissociation
of FSDDC-A independently from each other, and the effect of LPS was
TNF- dependent but not IL-1 dependent.
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, and LPS on FSDDC surface phenotype
Recently, we demonstrated that cells in FSDDC aggregates display a
surface phenotype similar to that of freshly obtained LC (17). Upon
subculture for 3 to 5 days, FSDDC-A release single cells with a
pronounced dendritic morphology and a surface phenotype analogous to
that of interdigitating DC (17). Dissociation of FSDDC-A with
IL-1, TNF-, or LPS also gave rise to cells that were highly
dendritic (see Fig. 1). To determine whether dissociation was also
accompanied by a change in surface phenotype, we performed multicolor
flow cytometry. Cells were stained with FITC-anti-CD45 and the
indicated mAb, and CD45+ cells (>90% of all cells
present) were selected for analysis. Treatment of FSDDC-A with IL-1,
IL-1ß, TNF-, and LPS for 18 h induced increased expression of
MHC class II Ag, CD40, CD80, and CD86 (Fig. 5). Effects on CD86 expression were
especially dramatic. Incubation of FSDDC-A with IL-6, IL-10, or
TGF-ß1 did not result in a change in FSDDC surface phenotype (data
not shown). Data presented in Fig. 6
demonstrate that treatment of FSDDC-A with IL-1ß or TNF- also
caused down-regulation of cell surface E-cadherin expression and that
down-modulation of E-cadherin and up-regulation of MHC class II Ag
occurred with similar time courses. Similar results were obtained after
stimulation with LPS (data not shown). These data, when considered in
conjunction with data presented in Fig. 2, indicate that loss of FSDDC
cell surface E-cadherin (Fig. 6) and decreased E-cadherin-mediated
adhesion (Fig. 2) are temporally linked.
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To determine whether cytokine-induced decreases in cell surface
E-cadherin levels reflected alterations in steady-state mRNA levels,
FSDDC E-cadherin mRNA levels were quantitated by Northern blotting.
Concentrations of IL-1ß and TNF- that down-regulated FSDDC cell
surface E-cadherin also decreased E-cadherin mRNA levels in a
dose-dependent fashion (Fig. 7A). Time course
studies revealed that IL-1ß and TNF- each rapidly down-regulated
FSDDC E-cadherin mRNA levels, with a 50% decrease occurring about
2 h after cytokine addition (Fig. 7B). Flow
cytometric data obtained in parallel (Fig. 6) demonstrated that
cytokine-induced changes in cell surface E-cadherin levels were minimal
at 2 h and became evident only considerably later. These results
suggest that IL-1 and TNF- cause a reduction of E-cadherin mRNA
steady-state levels leading to reduced E-cadherin surface expression,
which, in turn, results in a loss of E-cadherin-mediated adhesion and
dissociation of FSDDC-A.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and LPS in the
activation and mobilization of LC and other nonlymphoid DC from their
tissues of origin but did not allow mechanisms, or even targets, of
action of these inflammatory mediators to be precisely determined (7, 9, 10, 11, 12, 13, 14, 16). To facilitate studies of the biochemistry and cell biology
of LC/DC cell activation and maturation, as well as studies of
E-cadherin-mediated adhesion involving cells of the DC lineage, we
defined culture conditions that permit LC-like cells to be expanded
from murine fetal skin (17). LC from adult epidermis are not suitable
for these kinds of studies for several reasons. First, the spontaneous
activation and maturation that accompanies preparation of LC from
epidermis precluded in vitro studies of the LC-stimulatory properties
of proinflammatory cytokines. Second, routine isolation of
keratinocyte-free LC in quantities sufficient to permit the experiments
described in this report to be conducted was not possible. The present study demonstrates that inflammatory mediators that mobilize LC from epidermis act directly on LC-like DCs expanded from murine fetal skin (FSDDC) in vitro and regulate E-cadherin expression and function in these cells. Because these same stimuli did not influence keratinocyte E-cadherin expression (our unpublished observations), we conclude that cadherin expression and function in leukocytes and epithelial cells can be differentially regulated. Loss of E-cadherin-mediated adhesion in FSDDC was accompanied by the development of dendritic morphology, up-regulation of class II MHC Ag and costimulatory molecules, and decreased expression of E-cadherin on FSDDC surfaces. Activation of LC in vivo results in similar changes in surface phenotype within a similar time frame (7, 9, 14). Changes in cellular morphology and in surface phenotype did not occur in FSDDC that were disaggregated by mAb that blocked E-cadherin homophilic interaction (ECCD-1 (34)), indicating that loss of E-cadherin-mediated adhesion in aggregates is a consequence of activation and maturation of FSDDC and not a trigger of this process (our unpublished observations).
LPS treatment of FSDDC-A stimulated increases in steady-state levels of
mRNAs encoding IL-1, IL-1ß, and IL-1RA measured at 18 h,
while TNF- mRNA levels were comparable in unstimulated and
LPS-treated cells. Quantitation of proinflammatory cytokines in
supernatants revealed that FSDDC-A released small amounts of TNF-
spontaneously and considerably more after LPS treatment. Although
immunoreactive IL-1 was not detectable in supernatants conditioned by
unstimulated or LPS-stimulated FSDDC, significant amounts of IL-1
and IL-1ß were present in freeze-thaw lysates of LPS-treated cells.
IL-1 did not induce TNF- production, and TNF- did not stimulate
IL-1 release. Results of Ab inhibition studies were consistent with the
cytokine release data. The activity of LPS on E-cadherin-mediated
adhesion involving FSDDC was dependent on TNF- but not on IL-1.
Furthermore, anti-IL-1 reagents did not inhibit the action of
TNF-, and anti-TNF- mAb did not inhibit the action of
IL-1.
Northern blot analysis indicated that IL-1 and TNF- treatment of
FSDDC-A induced a rapid reduction in steady-state E-cadherin levels
that preceded decreases in cell surface E-cadherin. E-cadherin mRNA
levels decreased by 50% within 2 h after addition of IL-1 or
TNF-, while cell surface E-cadherin protein levels began to decrease
only several hours later. Subsequent changes in FSDDC E-cadherin levels
occurred with a time course compatible with the 5-h half-life
previously reported for completely processed E-cadherin in epithelial
cells (35). This implies that E-cadherin expression and function in
FSDDC may be primarily (or entirely) regulated at the mRNA level.
Currently, we cannot differentiate effects of IL-1 and TNF- on
E-cadherin transcription from those on mRNA stability. Based on prior
studies, regulation at the transcriptional level seems likely.
In the past several years, cis-acting elements that regulate
expression of murine and human E-cadherin genes have been identified
and significant homologies have been noted. Particular emphasis has
been placed on an upstream palindromic sequence termed E-pal, and E-pal
binding proteins have been demonstrated in epithelial as well as
mesenchymal cells. Deletion experiments suggest that E-pal binding
proteins act as transcriptional activators in epithelial cells and
repressors in mesenchymal cells, resulting in tissue-specific
expression of E-cadherin (36, 37). Other studies indicate that the
E-cadherin promoter is often silenced in carcinoma cells by
hypermethylation of GC-rich regions (38, 39). Overexpression of the
transmembrane tyrosine kinase-encoding oncogene ERBB2 has
been shown to decrease E-cadherin in mammary epithelial cells by
inhibiting E-cadherin transcription, but it is not clear whether this
effect is mediated through DNA methylation (40). Selective activation
of AP-1 transcription factors (c-fos and
c-jun) in mammary epithelial cells also results in
decreased E-cadherin expression and/or function (41, 42, 43). Because AP-1
is an important component of ERBB2, IL-1, TNF-, and LPS
signal transduction pathways, it is possible that increased expression
or activation of this transcription factor complex in FSDDC is
responsible for the down-regulation of E-cadherin expression that we
have observed. It will be of interest to determine whether any or all
of these pathways are activated in FSDDC by IL-1, TNF-, and LPS, or
whether FSDDC E-cadherin expression is controlled via novel regulatory
mechanisms.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Mark C. Udey, Dermatology Branch, National Cancer Institute, Building 10, Room 12N238, Bethesda, MD 20892.
3 Abbreviations used in this paper: DC, dendritic cell; LC, Langerhans cell; FSDDC, fetal skin-derived dendritic cell; FSDDC-A, fetal skin-derived dendritic cell aggregates; GM-CSF, granulocyte/macrophage colony-stimulating factor; MIF, macrophage migration inhibitory factor; CCD, cytochalasin D; GAPDH, glyceraldehyde-3-dehydrogenase.
Received for publication September 16, 1997. Accepted for publication December 19, 1997.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 K. Matthews, C. M. Leong, L. Baxter, E. Inglis, K. Yun, B. T. Backstrom, J. Doorbar, and M. Hibma Depletion of Langerhans Cells in Human Papillomavirus Type 16-Infected Skin Is Associated with E6-Mediated Down Regulation of E-Cadherin J. Virol., August 1, 2003; 77(15): 8378 - 8385. [Abstract] [Full Text] [PDF]
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 S. Nakae, Y. Komiyama, S. Narumi, K. Sudo, R. Horai, Y.-i. Tagawa, K. Sekikawa, K. Matsushima, M. Asano, and Y. Iwakura IL-1-induced tumor necrosis factor-{alpha} elicits inflammatory cell infiltration in the skin by inducing IFN-{gamma}-inducible protein 10 in the elicitation phase of the contact hypersensitivity response Int. Immunol., February 1, 2003; 15(2): 251 - 260. [Abstract] [Full Text] [PDF]
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 N. Carayol, I. Vachier, A. Campbell, L. Crampette, J. Bousquet, P. Godard, and P. Chanez Regulation of E-cadherin expression by dexamethasone and tumour necrosis factor-{alpha} in nasal epithelium Eur. Respir. J., December 1, 2002; 20(6): 1430 - 1436. [Abstract] [Full Text] [PDF]
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F. Geissmann, M.C. Dieu-Nosjean, C. Dezutter, J. Valladeau, S. Kayal, M. Leborgne, N. Brousse, S. Saeland, and J. Davoust Accumulation of Immature Langerhans Cells in Human Lymph Nodes Draining Chronically Inflamed Skin J. Exp. Med., August 19, 2002; 196(4): 417 - 430. [Abstract] [Full Text] [PDF]
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K. Seiffert, J. Hosoi, H. Torii, H. Ozawa, W. Ding, K. Campton, J. A. Wagner, and R. D. Granstein Catecholamines Inhibit the Antigen-Presenting Capability of Epidermal Langerhans Cells J. Immunol., June 15, 2002; 168(12): 6128 - 6135. [Abstract] [Full Text] [PDF]
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 N. Carayol, A. Campbell, I. Vachier, B. Mainprice, J. Bousquet, P. Godard, and P. Chanez Modulation of Cadherin and Catenins Expression by Tumor Necrosis Factor-alpha and Dexamethasone in Human Bronchial Epithelial Cells Am. J. Respir. Cell Mol. Biol., March 1, 2002; 26(3): 341 - 347. [Abstract] [Full Text] [PDF]
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G. M. Halliday and S. Le Transforming growth factor-{beta} produced by progressor tumors inhibits, while IL-10 produced by regressor tumors enhances, Langerhans cell migration from skin Int. Immunol., September 1, 2001; 13(9): 1147 - 1154. [Abstract] [Full Text] [PDF]
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K. Y. Vermaelen, I. Carro-Muino, B. N. Lambrecht, and R. A. Pauwels Specific Migratory Dendritic Cells Rapidly Transport Antigen from the Airways to the Thoracic Lymph Nodes J. Exp. Med., January 1, 2001; 193(1): 51 - 60. [Abstract] [Full Text] [PDF]
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 E. Riedl, J. Stockl, O. Majdic, C. Scheinecker, W. Knapp, and H. Strobl Ligation of E-cadherin on in vitro-generated immature Langerhans-type dendritic cells inhibits their maturation Blood, December 15, 2000; 96(13): 4276 - 4284. [Abstract] [Full Text] [PDF]
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Y. Kobayashi, M. Matsumoto, M. Kotani, and T. Makino Possible Involvement of Matrix Metalloproteinase-9 in Langerhans Cell Migration and Maturation J. Immunol., December 1, 1999; 163(11): 5989 - 5993. [Abstract] [Full Text] [PDF]
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F. Geissmann, P. Revy, A. Regnault, Y. Lepelletier, M. Dy, N. Brousse, S. Amigorena, O. Hermine, and A. Durandy TGF-{beta}1 Prevents the Noncognate Maturation of Human Dendritic Langerhans Cells J. Immunol., April 15, 1999; 162(8): 4567 - 4575. [Abstract] [Full Text] [PDF]
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H. Saeki, A. M. Moore, M. J. Brown, and S. T. Hwang Cutting Edge: Secondary Lymphoid-Tissue Chemokine (SLC) and CC Chemokine Receptor 7 (CCR7) Participate in the Emigration Pathway of Mature Dendritic Cells from the Skin to Regional Lymph Nodes J. Immunol., March 1, 1999; 162(5): 2472 - 2475. [Abstract] [Full Text] [PDF]
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B. Wang, L. Zhuang, H. Fujisawa, G. A. Shinder, C. Feliciani, G. M. Shivji, H. Suzuki, P. Amerio, P. Toto, and D. N. Sauder Enhanced Epidermal Langerhans Cell Migration in IL-10 Knockout Mice J. Immunol., January 1, 1999; 162(1): 277 - 283. [Abstract] [Full Text] [PDF]
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E. von Stebut, Y. Belkaid, T. Jakob, D. L. Sacks, and M. C. Udey Uptake of Leishmania major Amastigotes Results in Activation and Interleukin 12 Release from Murine Skin-derived Dendritic Cells: Implications for the Initiation of Anti-Leishmania Immunity J. Exp. Med., October 19, 1998; 188(8): 1547 - 1552. [Abstract] [Full Text] [PDF]
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T. Jakob, P. S. Walker, A. M. Krieg, M. C. Udey, and J. C. Vogel Activation of Cutaneous Dendritic Cells by CpG-Containing Oligodeoxynucleotides: A Role for Dendritic Cells in the Augmentation of Th1 Responses by Immunostimulatory DNA J. Immunol., September 15, 1998; 161(6): 3042 - 3049. [Abstract] [Full Text] [PDF]
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