From the Department of Dermatology and Department of
Morphology, Geneva University Medical School, 1211 Geneva 14, Switzerland and the § Institute of Biochemistry, University
of Lausanne, 1066 Epalinges, Switzerland
Received for publication, December 2, 2002
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ABSTRACT |
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Acquired Fas ligand (FasL)-mediated cytolytic
activity of human keratinocytes causes the massive keratinocyte cell
death that occurs during toxic epidermal necrolysis, a deadly adverse
drug eruption. Under normal conditions keratinocyte apoptosis is a rare
event in the epidermis although keratinocytes express the death
receptor Fas and its ligand. Here we have investigated why this is so.
We show that Fas, FasL, Fas-associated death domain, and caspase-8
mRNA are detectable in the epidermis, primary keratinocyte cultures, and keratinocyte cell line and that Fas protein is expressed in keratinocytes of all subcorneal layers of the epidermis, whereas FasL is only expressed in the basal and first suprabasal layers. Coexpression of Fas and FasL therefore occurs in basal and suprabasal keratinocytes. In vitro, keratinocytes are killed by
recombinant FasL in a dose-dependent manner, but they are
unable to kill Fas-sensitive target cells despite FasL expression.
Analysis of keratinocyte culture supernatants and treatment of
keratinocytes with metalloproteinase inhibitors excluded cell surface
expression of FasL and rapid metalloproteinase-mediated cleavage of
cell surface FasL. Fluorescence-activated cell sorter, confocal
microscopical, and electron microscopical analysis revealed that
keratinocyte FasL is localized intracellularly predominantly associated
to intermediate filaments. These data suggest that the observed
inability of keratinocyte FasL to induce apoptosis under physiological
conditions is due to its cellular localization and also indicate that
intermediate filaments may be involved in regulating the subcellular
localization of FasL.
FasL1 is a member of the
tumor necrosis factor protein superfamily. It is a type II
membrane protein lacking a signal sequence, but having an internal
hydrophobic domain that allows membrane anchorage of the ligand (1).
Upon contact with FasL, cells expressing Fas rapidly undergo apoptosis
(2, 3) by way of activation of an intracellular signaling pathway. This
pathway involves a distinct cytoplasmic motif called the "death
domain" (4, 5) that interacts with a protein called FADD (6, 7). The
recruitment of FADD allows the connection of the Fas receptor to the
cysteine protease caspase 8 (ICE-like cysteine proteases) (FLICE/MACH)
(8, 9).
The Fas system has proven to be essential in contributing to the
functional integrity of the epidermis. Recent evidence has shown that
Fas-induced keratinocyte apoptosis in response to UV prevents the
accumulation of procarcinogenic p53 mutations by deleting UV-mutated
keratinocytes (10). Furthermore, strong evidence exists that
dysregulation of Fas expression and/or signaling contributes to the
pathogenesis of diseases such as toxic epidermal necrolysis (11) and
acute cutaneous graft versus host disease (12, 13).
Immunohistochemical studies of normal human skin have reported the
expression of Fas on keratinocytes (14-17) and shown that agonistic
anti-Fas antibody can mediate apoptosis of interferon- Cell Culture--
Interfollicular epidermal keratinocytes (HEK)
isolated from human epidermis and outer root sheath keratinocytes (ORS)
isolated from human hair follicles (22) were maintained in KGM medium (Bioreba, Reinach, Switzerland) (basal culture conditions). The Hacat
cell line (spontaneously immortalized keratinocyte cell line) was
obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained in DMEM medium (Invitrogen,
Paisley, UK) supplemented with 10% FCS, 100 µg/ml streptomycin
sulfate, 100 IU/ml penicillin. Fas-sensitive A20, and Fas-resistant
A20R mouse B cell lymphoma cells, were maintained in DMEM medium
(Invitrogen) supplemented with 10% FCS, 100 µg/ml streptomycin
sulfate, 100 IU/ml penicillin, 0.01 mM RNase Protection Assay--
RNase protection assays were
performed using 15 µg of total RNA with the RiboQuant multi-probe
RNase protection assay system (Pharmingen), according to the
manufacturer's instructions. In summary, RNA was hybridized overnight
with the in vitro translated 32P-labeled probe
(hAPO-3 multiprobe template, Pharmingen), and samples were then
treated with RNase A and proteinase K, phenol/chloroform-extracted, and
precipitated with ethanol. The protected fragments were resolved on a
5% acrylamide/urea gel and further analyzed by autoradiography.
Reverse Transcription-Polymerase Chain Reaction
(RT-PCR)--
Reverse transcription and amplification were performed
from total RNA using the Qiagen One-step RT-PCR kit (Qiagen Ag, Basel, Switzerland). RT-PCR for FasL was carried out using the following forward and reverse primers 5'-CTC TGG AAT GGG AAG ACA CC-3' and 5'-ACC
AGA GAG AGC TCA GAT ACG-3'. For Immunohistochemistry and
Immunofluorescence--
Immunohistochemistry was performed on
cryosection as described (23) using UB2 anti-Fas antibody (Immunotech,
Marseille, France) and isotype-matched control antibody (mouse IgG1).
For immunofluorescence, 6-µm-thick cryostat sections were air-dried,
fixed for 10 min with ice-cooled acetone/ethanol (1:1), and rehydrated
with PBS. Sections were then incubated at room temperature for 60 min
with anti FasL rat IgM antibody A-11 (Alexis, San Diego, CA) and with
anti- FACS Staining--
FACS staining was performed on HaCaT cells
permeabilized or not by 1% formaldehyde in PBS. Cells were then
incubated at 4 °C for 1 h in FACS buffer (PBS, 5% FCS, plus
1% saponin for permeabilized cells) containing 1 µg of mouse
anti-FasL G247-4 antibody (PharMingen) or 1 µg of the
isotype-matched control MOPC-21 (PharMingen). After two washings
in FACS buffer, cells were stained at 4 °C for 45 min with 1 µg of
Alexa 488-conjugated anti-mouse (Molecular Probes, Inc., Eugene, OR).
After two washings, cells were directly analyzed on a
FACScanTM (BD Biosciences) using Cellquest 3.0 software. For FACS analysis in non-permeabilized cells, PI
exclusion was used to eliminate dead cells.
Fas and FasL Functionality Assays--
For analysis of Fas death
signaling function, cells were seeded in 96-well microtiter plates at a
density of 104 cells per well. After adhesion, serial
concentrations of recombinant soluble FLAG-tagged FasL (Alexis, San
Diego, CA) plus enhancer (anti-FLAG Ab, Alexis) at 1 µg/ml
concentration were added. Viability of cells was determined 24 h
later by using the Boehringer cell proliferation reagent WST-1
according to the manufacturer's instructions (Boehringer GmbH,
Mannheim, Germany). Percentage of cell viability was determined against
control untreated cells.
Cytolytic FasL function of cultured keratinocytes was determined using
a 20-h 125I-deoxyuridine release assay. A20 and A20R target
cells were labeled with 1mCi of 125I-deoxyuridine (Amersham
Biosciences) for 2 h at 37 °C in a humidified 5%
CO2 incubator and then washed three times in medium.
Labeled target cells were seeded at 104 cells per well with
keratinocytes at the indicated effector/target cells ratios in flat
bottom 96-well microtiter plates. After incubation for 20 h at
37 °C, 125I-deoxyuridine-labeled fragmented DNA released
in culture medium was pooled with cytoplasmic
125I-deoxyuridine-labeled fragmented DNA recovered by cell
lysis. Pooled supernatants were assayed for radioactivity using a
Cytolytic function of FasL in human epidermis was determined as
described previously (11). Briefly, 600,000 Jurkat cells (in 100 µl
of medium) were overlaid on human skin cryosections and incubated for
6 h at 37 °C. Jurkat cell apoptosis was subsequently assessed
on 200,000 recovered cells by FACS analysis using an annexin-V assay
(PharMingen). Jurkat cells overlaid on 293 and 293-FasL
(previously cultured on coverslips) for 6 h were used as negative
and positive controls, respectively.
Immunoprecipitation--
To identify soluble FasL in the
supernatant of 293-WT, 293-FasL, and HEK cells, cells were cultured in
appropriate medium containing 230 µCi/ml of 35S-Cyst/Met
(Amersham Biosciences) at 37 °C for 24 h, and supernatants were
collected. After preclearing for 5 h with protein A, supernatants were incubated with 10 µg of Fas-Fc or CD44-Fc (control) for 2 h. Protein A was then added and the incubation of supernatants was
continued overnight at 4 °C. After three washings with buffer (0.5%-Triton X-100, 20 mM Tris-HCl, pH 7.6, 150 mM-NaCl), material bound to protein A was eluted with
electrophoresis sample buffer containing 50 µM
dithiothreitol and subjected to SDS-PAGE. For fluorography, the gel was
incubated with Amplify (Amersham Biosciences) for 30 min, dried, and
exposed to x-ray film at Electron Microscopy--
After informed consent of the patients
and authorization of the institutional ethical committee for clinical
investigation, keratome samples of control human skin were obtained
from specimens of abdominal reduction surgery.
Immediately after sampling, fragments were fixed at room temperature in
a 0.1% glutaraldehyde, 4% paraformaldehyde solution for 5 min and
then exposed for 55 min to 4% paraformaldehyde. The fragments were
then embedded at low temperature in Lowicryl K4M resin and
thin-sectioned using a Reichert OM10 ultramicrotome. Sections were
first incubated 10 min in PBS containing 0.5% bovine serum albumin and
then exposed at room temperature for 2 h to undiluted anti-FasL
mouse IgG1 monoclonal antibody NOK-1 (PharMingen International).
After rinsing, the sections were exposed 1 h at room temperature
to a preparation of 15-nm gold particles, coated with protein A, and
diluted 1:200. Sections were stained 10 min with 2% uranyl acetate and
examined in a Philips CM10 electron microscope.
Negative controls included 1) skin sections incubated with undiluted
isotype matched control antibody MOPC-21 (PharMingen International), followed by protein A-coated gold particles; 2) skin sections incubated with protein A-gold particles only; 3) wild
type 293 cells (not expressing human FasL) incubated with the NOK-1
monoclonal antibody followed by protein A-coated gold particles. None
of these controls resulted in a specific staining (not shown). Positive
controls were provided by incubations of 293 cells that were stably
transfected for human Fas ligand, with the NOK-1 monoclonal antibodies,
followed by protein A-coated gold particles. These incubations resulted
in a clear cytoplasmic labeling of the transfected cells (not shown).
Evaluation of labeling distribution was performed by taking photographs
of about 40 keratinocytes, in the suprabasal layers of epidermis of
three skin samples, at a constant 60,000-fold magnification. Gold
particles were scored in each photograph (representing each a surface
of 7.93 µm2) and attributed to one of the following
compartments: junctional membrane, non-junctional membrane
(desmosomes), cytosol, lysosomes, and other vesicles (Golgi apparatus
and endoplasmic reticulum, whose membranes are unsatisfactorily
preserved after K4M embedding, were not scored separately and were
included in the "vesicle" counts), intermediate filaments,
mitochondria, and nucleus. Values were calculated as number of protein
A-coated gold particles per 100 µm2 of cytoplasm and
compared between groups by analysis of variance and Student's unpaired
t tests, using the Statistical Package for Social Sciences
(SPSS Inc., Chicago).
The Fas Death-signaling Pathway Is Functional in Human
Keratinocytes Cultured under Basal Conditions--
RNase protection
was performed for Fas, FADD, and caspase 8 using total RNA extracted
from whole skin, split dermis and epidermis, primary keratinocyte
cultures (HEK, interfollicular epidermal keratinocytes and ORS, outer
root sheath keratinocytes). As FasL was not detectable by RNase
protection (data not shown), we used the more sensitive RT-PCR
technique to analyze FasL mRNA expression as indicated in the next
result paragraph. As shown in Fig.
1a, specific protected
fragments for Fas, FADD, and caspase 8 were detected in each RNA
sample, demonstrating that key initial effectors of the Fas signaling
pathway are expressed by human keratinocytes, both in vitro
and in vivo. Immunohistochemical staining revealed, as
reported previously, a membrane pattern of Fas staining in all
subcorneal layers of the epidermis (Fig. 1b). To investigate whether Fas signaling pathway is functional in human keratinocytes, we
tested the ability of recombinant soluble FasL (rFasL) to induce apoptosis of immortalized HaCaT and primary HEK cells under basal culture conditions. As shown in Fig. 1c, under these
conditions, recombinant human FasL (rFasL) induced
dose-dependent death of Fas-sensitive Jurkat cells and
HaCaT and HEK cells, HaCaT and HEK cells being, however, less sensitive
(IC50 = 89 and 232 ng/ml, respectively) to rFasL than
Jurkat cells (IC50 = 25 ng/ml).
FasL Is Expressed in the Basal and Suprabasal Layers of Human
Epidermis--
FasL mRNA expression was investigated by RT-PCR in
the same total RNA samples previously used to analyze Fas expression by RNase protection assay. Indeed, RNase protection that is less sensitive
than RT-PCR technique did not allow detection of FasL mRNA. As
shown in Fig. 2a, a 381-bp
FasL fragment was amplified in all samples, demonstrating mRNA FasL
expression in keratinocytes of normal human skin. To investigate
whether FasL mRNA is translated into protein in human skin, we
analyzed FasL distribution by immunofluorescence in tissue sections of
normal human skin. As shown in Fig. 2b, strong FasL
expression, consistent with RT-PCR results, was detected in epidermal
keratinocytes. Keratinocyte FasL protein was confined to basal and
first suprabasal layers of the epidermis, homogeneously distributed
within the cytoplasm and without plasma membrane enhancement. Specificity of the immunolabeling was established using isotype-matched control antibody (Fig. 2b, right panel).
Keratinocyte FasL Is Not Capable of Inducing Cell Death in Vitro
and ex Vivo--
The above experiments demonstrate that Fas and FasL
are coexpressed in the lower epidermis and that keratinocyte Fas death signaling pathway is functional under basal culture conditions. To
determine whether keratinocyte FasL is cytolytically active and able to
induce apoptosis in Fas-expressing keratinocytes, we analyzed the
capacity of primary HEK to induce Fas-mediated cell death. When
keratinocytes were incubated with radioactively labeled A20
(Fas-sensitive) or A20R (Fas-resistant) cells at various effector:target cell ratios, no significant level of apoptotic cell
death could be detected in A20 cells after coincubation with HEK. Thus
under tested conditions keratinocyte FasL is non-cytolytic. This result
was confirmed by the inability of human skin samples to induce
apoptosis of Fas sensitive Jurkat cells (Fig.
3b). The low percentage of
apoptotic cells detected (5-10%) in both experiments could not be
decreased by incubation with Fas-Fc fusion protein, demonstrating that
keratinocyte FasL is indeed non-lytic in vitro and ex
vivo.
Absence of Surface FasL in Human Keratinocytes in Vitro--
To
investigate whether the lack of keratinocyte cytolytic activity was due
to the absence of FasL at keratinocyte membrane, we analyzed FasL
expression at the surface of HEK cells by FACS. As shown in Fig.
4a, HEK cells were clearly
negative for membrane FasL (upper left). To investigate whether the
absence of membrane FasL on keratinocytes could be due to proteolytic
cleavage and release of surface FasL into the culture medium, we
performed an immunoprecipitation of conditioned culture medium with a
Fas-Fc fusion protein. As shown in Fig. 4b,
immunoprecipitation of cell culture supernatant from
293-FasL-transfected cells revealed significant levels of soluble FasL
at 25 kDa, whereas culture supernatants from wild type 293 and primary
cultures of HEK did not. These data suggest that FasL protein may not
be translocated to the membrane of keratinocytes and may be retained
within their cytoplasm. This hypothesis was confirmed by FACS analysis
performed on permeabilized HEK cells (Fig. 4a, upper
right), showing a marked shift in fluorescence intensity (35% of
cells) in comparison to non-permeabilized cells and permeabilized cells
incubated with an isotype-matched control antibody. The marked shift
between both antibodies correspond to specific binding as it was
cancelled by incubation of anti-FasL G247-4 antibody by a 5-fold excess
of recombinant FasL protein (data not shown). These data point out that
FasL is localized intracellularly in human HEK cells.
Keratinocyte FasL Is Cytoplasmic in Vivo--
To establish the
in vivo localization of FasL in keratinocytes, normal human
skin was immunostained both with anti-FasL and anti-integrin mAbs and
analyzed by confocal microscopy (Fig.
5a). This double labeling
revealed that cellular localizations of keratinocyte FasL
(green staining) and membrane specific integrin
(red staining) were distinct although some little
colocalization occurred in suprabasal epidermal layers (Fig.
5a, yellow). The same experiment performed in
cultured human keratinocytes HEK (Fig. 5, b and
d) confirmed the predominant cytoplasmic localization of
FasL. These data show that keratinocyte FasL is predominantly located
inside the cytoplasm both in vitro and in
vivo.
FasL Is Associated with Intermediate Filaments in
Keratinocytes--
Using anti-FasL antibody (NOK-1) and protein
A-coated gold particles for immunoelectron microscopical analysis of
human skin, FasL labeling was detected in most living keratinocytes of
human interfollicular epidermis in agreement with the previously shown molecular, biochemical, and immunohistochemical data. In the plastic- and low temperature-embedded material, which we used, this labeling was
almost exclusively restricted to the cytoplasm of keratinocytes (Fig.
6, a and b). The
labeling of keratinocytes was considered specific in view of the
various controls (see "Experimental Procedures," including the
observation that labeling was absent when using the isotypic irrelevant
antibody MOPC-21) (Fig. 6c). Quantitative evaluation further
showed that different organelles of keratinocytes were not similarly
labeled under the experimental conditions that elicited a sizeable
staining of these cells for FasL. FasL was predominantly observed in
the cytosol and intermediate filament compartments (total number of
gold particles per 100 µm2 of these compartments was
113.1 ± 15.8, n = 39 and 105.4 ± 14.2, n = 39, respectively) (Table
I), irrespective of whether these organelles were located close to (Fig. 6a) or at distance
from the cell membrane (Fig. 6b) and also to some extent in
lysosomes and vesicles (22.9 ± 6.8, n = 39). In
contrast, several other organelles, including desmosomal and
non-junctional regions of the cell membrane, mitochondria (Fig.
6b), and nucleus failed to show any specific labeling above
background levels (Table I).
Several studies have shown that Fas-mediated apoptosis is
important for the maintenance of epidermal homeostasis (for review, see
Ref. 25). This is best illustrated by the fact that mice with mutant
non-functional FasL are significantly more resistant to UVB-induced
keratinocyte apoptosis ("sunburn cells") and, as a consequence,
accumulates UV-induced p53 mutations significantly more rapidly than
control mice (10). It is therefore very likely that keratinocyte FasL
expression and intact Fas signaling pathway in keratinocytes are
crucial for the prevention of UV induced skin cancer. Although Fas is
known to be expressed in human keratinocytes, and capable of signaling
death when triggered in the presence of interferon- Previous studies on human keratinocytes have shown FasL expression at
the mRNA and protein levels in vitro (18, 20, 21). Our
data confirm these observations as they show constitutive keratinocyte
FasL expression in vitro and additionally show constitutive in vivo expression of FasL at the protein level in lower
human epidermis. FasL protein expression therefore colocalizes with Fas
expression at this location. In addition, our data demonstrate that the
Fas death signaling pathway is functional in resting keratinocytes,
even without interferon- We provide here first evidence for an intracellular localization of
keratinocyte FasL. Indeed, FasL was clearly detected by FACS in
permeabilized but not in non-permeabilized keratinocytes and was shown
to be essentially localized in the cytoplasm of keratinocytes in
vitro and in vivo, using confocal and electron microscopical analysis. Maintenance of keratinocyte FasL in an intracellular localization may therefore be a physiological means by
which to guarantee a rapid availability of cell surface FasL if
required, while keeping this protein out of reach of cell surface Fas
that is constitutively expressed and functional in keratinocytes. In
this way, keratinocytes can protect themselves from spontaneous Fas-mediated apoptosis under physiological situations despite harboring
all the proteins necessary to rapidly trigger it. In this way,
keratinocytes can also suicide themselves under stress situations. Indeed, one can hypothesize that in a situation such a
harmful UVB exposition of skin, intracellular FasL can be translocated to the keratinocyte cell surface where Fas receptor is present and then
lead to apoptotic destruction of UVB-damaged keratinocytes and then
avoid accumulation of mutated cells in skin. Such an exclusive
intracellular localization of FasL has also been shown for cytotoxic T
lymphocytes, in which FasL is stored in secretory lysosomes and
exocytosed following T cell receptor engagement (29).
Using immunoelectron microscopy we have further analyzed the
subcellular localization of FasL and shown that this protein is
predominantly associated with intermediate filaments, the remainder being localized to lysosomes and cytoplasmic vesicles. Our observations suggest that FasL may be docking to intermediate filaments, even though
the molecular interaction between FasL and intermediate filaments
remain to be elucidated. Thus it is possible that intermediate filaments of human keratinocytes are somehow involved in regulating the
transport of FasL to the plasma membrane. Intermediate filaments have
already been involved in the translocation of proteins to the cell
surface in response to certain stimuli. Indeed, in 3T3-L1 adipocytes
disruption of intermediate filaments has been shown to induce marked
dispersion of the perinuclear GLUT4 sugar transporter to peripheral
regions of the cells (30). Further investigation of the association
between FasL and keratins may help explain how keratinocyte FasL can
become cytolytic under selected circumstances and contribute to the
pathogenesis of skin diseases such as toxic epidermal necrolysis
(11).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
pretreated
cultured keratinocytes (15-17). To date, only two studies have
reported FasL protein expression in the epidermis in vivo (11, 18). Several studies have however shown FasL protein in cultured
keratinocytes by Western blot analysis (18-21). The exact cellular
localization and the cytolytic potential of keratinocyte FasL are not
yet known under normal conditions. Here we investigated this in the aim
of better understanding how the cytolytic activity of keratinocyte FasL
is regulated.
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mercaptoethanol,
and 15 mM Hepes. 293 human embryonic kidney cells (293-WT),
293 stably transfected with human FasL (293-FasL) and HpK1a
keratinocytes were maintained in DMEM medium (Invitrogen) supplemented
with 10% FCS, 100 µg/ml streptomycin sulfate, 100 IU/ml penicillin.
-actin 5'-TGA TGG ACT CCG GTG ACG
G-3' and 5'-TGT CAC GCA CGA TTT CCC GC-3'. PCR products (40 cycles) of
FasL (381 bp, intron spanning) and
-actin (179 bp) were analyzed by
1.5% agarose gel electrophoresis.
1-integrin mouse IgG antibody AJ2 (kindly provided by C. E. Klein, University of Würzburg, Würzburg, Germany)
(24), diluted 1:60 and 1:40 in PBS supplemented with 1% bovine serum
albumin, respectively. For negative control, sections were incubated
with rat IgM and mouse IgG, respectively. Sections were then revealed
with fluorescein isothiocyanate-conjugated mouse anti rat IgM diluted
1:200 or with Texas red-conjugated anti mouse IgG diluted 1:200
(Jackson ImmunoResearch, West Grove, PE). Results were routinely
analyzed under fluorescence microscope. Some experiments were analyzed
using a confocal laser-scanning microscope, Zeiss LSM 410 invert,
equipped with argon (488 nm) and HeNe (543 nm) lasers.
-counter. Percentage of apoptosis was calculated as: 100 × (sample 125I release
spontaneous 125I
release)/(maximum 125I release
spontaneous
125I release).
80 °C for 3 days.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Human keratinocyte expression and function of
molecules implicated in upstream signaling of Fas-mediated cell
death. a, RNase protection analysis of Fas (top
row), FADD (middle row), and caspase 8 (bottom
row) mRNA in whole human skin, split dermis, and epidermis,
primary keratinocyte cultures (HEK and ORS), and HaCaT keratinocyte
cell line (upper panels). Equal RNA loading was assessed by
RNase protection analysis of glyceraldehyde-3-phosphate dehydrogenase
(GapdH) mRNA (lower panel). b,
immunohistochemical staining of human skin cryosections using UB2
anti-Fas antibody (left panel) or isotype-matched control
antibody (right panel). c, viability assay of
HaCaT, HEK, and Jurkat cells after 24-h exposure to increasing
concentrations of recombinant soluble FasL.
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Fig. 2.
FasL expression in human keratinocytes
in vitro and in vivo.
a, RT-PCR analysis of FasL (top row) mRNA in
whole human skin, split dermis and epidermis, primary keratinocyte
cultures (HEK and ORS) and keratinocyte cell line (HaCaT) (upper
panel). Equal RNA loading was assessed by RT-PCR analysis of actin
mRNA (lower panel). b, immunofluorescence
staining of human skin cryosections using A11 anti-FasL antibody
(left panel) or isotype-matched control antibody
(right panel).
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Fig. 3.
Analysis of the cytolytic potential of
keratinocyte FasL under resting conditions in vitro
and in vivo. a,
125I-deoxyuridine release assay of A20 and A20R target
cells cocultured with human keratinocytes at the indicated
target:effector ratios for 20 h. A20 cells incubated with
recombinant soluble FasL are shown as a positive control. b,
apoptosis analysis by annexin-V test of Jurkat cells overlaid for
6 h on normal human skin cryosections preincubated
(Fas-Fc) or not (Cont) with Fas-Fc fusion
protein. Jurkat cells cultured in medium alone were used as control for
Jurkat cell viability (Cont). Jurkat cells overlaid for
6 h on 293 or 293-FasL cells are shown as negative and positive
controls, respectively.
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Fig. 4.
Analysis of the localization of FasL in
cultured keratinocytes. a, FACS analysis of cell
surface (non-permeabilized cells, upper left) or
intracellular (permeabilized cells, upper right) FasL
expression in HEK cells using G247-4 anti-FasL antibody (black
line) or an isotype-matched control antibody (gray
histogram). 293-WT and 293-FasL transfected cells were used as
negative and positive controls, respectively. b, analysis of
HEK culture supernatants for the presence of soluble FasL by
immunoprecipitation using Fas-Fc fusion protein. 293-WT and 293-FasL
culture supernatants were used as negative and positive controls,
respectively, and CD44-Fc as a control Fc-fusion molecule.
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Fig. 5.
Confocal microscopical analysis of FasL
localization in human keratinocytes in vitro and
in vivo. Confocal analysis of human skin
cryosections (a) and HEK cells (b) co-labeled
using immunofluorescence for FasL (green) and 1-integrin
(red). Yellow staining indicates colocalization.
Note that the great majority of FasL staining is intracellular. In
a, the dashed line illustrates the dermoepidermal
junction, whereas De. and CL. annotations
indicate dermis and corneal layer of the epidermis, respectively. 293T
cells transiently transfected with a human FasL cDNA were used as
controls for cell surface FasL staining: non-permeabilized cells
labeled with anti-FasL antibody showed a surface staining (c
and d), whereas no staining is observed in these cells with
an antibody directed against intracellular FLIP protein (e
and f).
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Fig. 6.
Electron microscopical analysis of the
subcellular localization of keratinocyte FasL.
a, Fas ligand immunolabeling is detected with NOK-1
antibody in several cytoplasmic compartments of human interfollicular
epidermal keratinocytes. Specific labeling was mostly found over
cytosol (gold particles circled in black) and
intermediate filaments (gold particles circled in
white), but was not above background level in other
organelles, including desmosomal regions of the cell membrane (portions
of two adjacent keratinocytes are seen in this large view).
b, the cytosolic and intermediate filament-associated
labeling is also observed in regions of the cytoplasm of spinous
keratinocytes, which were distant from the cell membrane and contained
unstained organelles, such as mitochondria. c, incubation of
adjacent sections from the very same skin sample with an isotypic but
irrelevant antibody (MOPC-21) did not result in a significant labeling
of human keratinocytes (the single gold particle visible in this field
is circled). The bar represents 300 nm in
a and c and 335 nm in b.
Distribution of FasL immunolabeling in human keratinocytes
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
(17), little is
known about the expression, cellular localization, and cytolytic
function of FasL in resting human keratinocytes in vitro and
in vivo. The present report analyzes for the first time the
exact cellular localization of keratinocyte FasL, and provides evidence
that FasL in resting keratinocytes has no cytolytic activity because of
its intracellular localization.
pretreatment, contrary to what was
suggested by a previous report (17). Coexpression of functional Fas and
FasL set the conditions for spontaneous Fas-mediated cell death of
keratinocytes in the basal and first suprabasal layers of the
epidermis. However, spontaneous keratinocyte apoptosis in these layers
is very seldom observed under physiological conditions (26). We
therefore investigated the cytolytic capacity of FasL expressed by
normal human keratinocytes in vitro and ex vivo.
Our data clearly show that keratinocyte FasL does not
induce apoptosis of either adjacent keratinocytes in culture or
Fas-sensitive cells overlaid on sections of normal human skin. This
lacking of cytolytic activity is probably due to the absence of FasL at the surface of keratinocyte cells, as demonstrated by FACS analysis. We
cannot exclude that lack of FasL surface expression could be due to the
weak expression of FasL. Indeed, we have unpublished data2 that showed cell
surface expression of FasL in cells treated to increase FasL mRNA
transcription level. However, previous reports in other cell types,
including activated T cells, have shown that the levels of FasL
expressed at the cell surface can be regulated by rapid
metalloproteinase cleavage of cell surface FasL, despite stable FasL
gene expression (27, 28). In these cell types, treatment with
inhibitors of matrix metalloproteinases specifically led to the
accumulation of cell surface FasL and to a decrease in soluble FasL in
the culture medium. Thus, we investigated whether the lack of membrane
FasL in normal human keratinocytes was due to excessive metalloprotease
cleavage. This is most likely not the case, since soluble FasL could
not be detected in culture supernatants of keratinocytes and
furthermore, since treatment of keratinocytes with metalloproteinase
inhibitors did not cause an increase in cell surface FasL staining, as
evaluated by FACS (data not shown).
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge G. Radlgruber and E. Suter for the excellent technical assistance and M. Pisteur for photographical assistance.
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FOOTNOTES |
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* This work was supported by grants of the Swiss National Science Foundation, the Ligue Genevoise contre le Cancer, the Fondation Leenaards, and the Ernst and Lucie Schmidheiny Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Dermatology, Geneva University Medical School, 1211 Geneva 14, Switzerland. Tel.: 41-22-7025811; Fax: 41-22-7025802; E-mail: Lars.French@medecine.unige.ch.
Published, JBC Papers in Press, December 7, 2002, DOI 10.1074/jbc.M212188200
2 I. Viard-Leveugle, R. R. Bullani, J. Schrenzel, J.-H. Saurat, J. Tschopp, and L. E. French, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: FasL, Fas ligand; FADD, Fas-associated death domain; FLICE, FADD-like ICE (caspase 8); ORS, outer root sheath keratinocytes; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; RT, reverse transcription; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorter; Ab, antibody; mAb, monoclonal antibody.
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