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*
Institute of Medical Immunology and
Department of Dermatology, Charité Campus Mitte, Humboldt University, Berlin, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, IFN-
, and IL-15,
respectively, increased significantly their intracellular as well as
cell surface-bound FasL expression in a time- and dose-dependent
manner. This cytokine-induced FasL expression was dependent on new
protein synthesis. Despite enhanced expression of cell surface-bound
FasL, no release of soluble FasL was measured in the cell supernatants
determined by ELISA. Stimulation of the cells with IL-6, IL-10, IL-12,
TGF-ß1, and GM-CSF did not modulate the constitutive FasL expression,
but IFN--mediated FasL up-regulation was significantly diminished by
IL-10 and TGF-ß1, respectively. Up-regulation of FasL on
IFN--stimulated keratinocytes led to increased apoptosis within
monolayers cultured for 48 h. Moreover, coculture experiments
performed with Fas+ Jurkat T cells revealed that enhanced
FasL expression on IFN--stimulated keratinocytes induced apoptosis
in cocultured T cells, demonstrating that up-regulated FasL was
functionally active. In summary, our data suggest the important
regulatory role of cytokine-controlled Fas/FasL interaction in the
cross-talk between keratinocytes and skin-infiltrating T cells for
maintenance of homeostasis in inflammatory skin
processes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, which further up-regulated Fas
expression on keratinocytes, were sensitive to Fas-mediated apoptosis
(22). Furthermore, UV irradiation of human keratinocytes increased both
Fas- and FasL cell surface expression, which led to UV-induced
apoptosis of IFN--treated keratinocytes (10, 23). Berthou et al.
(24) very recently described up-regulation of functional FasL on
keratinocytes following in vitro cultivation. However, up to now,
little information is available with regard to the regulatory
mechanisms controlling FasL expression on epithelial cells. It is well
established that UV light induces different cytokines (25, 26, 27).
Moreover, cytokines are involved in inflammatory cutaneous processes.
Therefore, we studied whether the expression of FasL on human
keratinocytes is directly regulated via cytokines released by
keratinocytes themselves or by skin-infiltrating immune competent
cells. Our data demonstrate that the proinflammatory cytokines IL-1ß,
IL-15, IFN-, and TNF- up-regulated FasL expression on
keratinocytes, whereas the anti-inflammatory cytokines IL-10 and
TGF-ß1 counteracted this effect. FasL expressed on
IFN--activated keratinocytes was functionally active, suggesting the
existence of a physiological mechanism leading to prevention of
uncontrolled proliferation of keratinocytes as well as unlimited
cytotoxicity against epidermal cells mediated by skin-infiltrating T
cells. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The following reagents were purchased: Ca ionophore A23187, PMA,
actinomycin D, and human rIL-1ß (Sigma, Deisenhofen, Germany); human
rIFN-, rTGF-ß1, and rIL-15 (Genzyme, Cambridge, MA); human rIL-6,
rIL-10, rIL-12, and rGM-CSF (R&D Systems, Wiesbaden-Nordenstadt,
Germany); human rTNF- (Bayer, Leverkusen, Germany); in situ cell
death detection kit-fluorescein (Boehringer Mannheim, Mannheim,
Germany); mouse anti-human Fas Ag mAb (F22120) and mouse
anti-human FasL Ag mAb (F37720; Transduction Laboratories,
Lexington, U.K.); mouse anti-human Fas mAb (clone CH-11, IgM;
Upstate Biotechnology, Lake Placid, NY), FITC-labeled mouse
anti-human FasL mAb (clone H11), recombinant soluble human
Apo-1/Fas ligand (522-001-C005), recombinant human Fas:Fc-IgG
(522-002-C050), and FITC-annexin V (209-250-T300; Alexis, Grunberg,
Germany); HRP-labeled sheep anti-mouse IgG (Seramun, Berlin,
Germany); mouse anti-human fibroblast mAb (clone ASO2),
FITC-labeled goat anti-mouse IgG (H+L) mAb, FITC-labeled (33024X),
and unlabeled mouse IgG1 mAb (33021A; Dianova, Hamburg, Germany); mouse
anti-human CD3 mAb (clone 145-2C11; PharMingen, San Diego, CA); and
sFas ligand ELISA (5255, Coulter-Immunotech Diagnostics, Hamburg,
Germany). If not stated otherwise all media and supplements were
obtained from Life Technologies Europe (Karlsruhe, Germany). All fine
chemicals were supplied by Sigma.
Cells and cell culture
Human epidermal cell suspensions were obtained from normal donors undergoing foreskin surgery. Freshly isolated epidermal cells were obtained by trypsin and were cultured in serum-free medium (KBM, BioWhittaker, Heidelberg, Germany) with full supplements (0.1 ng/ml human epidermal growth factor, 0.5 µg/ml hydrocortisone, 5 µg/ml insulin, 7.5 mg/ml bovine pituitary extract, 50 µg/ml gentamicin, 50 ng/ml amphotericin, and 0.15 mM calcium) according to the manufacturer’s instruction. For the performed experiments cells were derived from the fourth to sixth passages grown as a monolayer to subconfluence. Contamination of the keratinocyte culture by fibroblasts was always <0.5% as determined routinely by flow cytometry using anti-human fibroblast mAb (clone ASO2). The human keratinocyte cell line HaCaT was cultured in DMEM supplemented with 5% (v/v) FCS and 1% (v/v) antibiotic/antimycotic solution (28). Cells of the human A549 pulmonary epithelial cell line that show features of type II alveolar epithelial cells (29) were cultured in FK12 medium, containing 10% (v/v) FCS, 1% (v/v) antibiotic/antimycotic solution, and 20 mM sodium hydrogen carbonate. The human T cell leukemia cell line Jurkat was grown in RPMI 1640 (10% (v/v) FCS and 1% (v/v) antibiotic/antimycotic solution). All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. Only cell preparations with a viability >95%, analyzed by trypan blue exclusion test, were used for the experiments.
Cytokine stimulation experiments
When keratinocytes became semiconfluent, they were washed and
incubated with the given cytokines in a volume of 1 ml for 24 h.
In some experiments cells were preincubated for 24 h with TGF-ß1
(100 ng/ml) or IL-10 (100 ng/ml). Thereafter, cells were washed twice,
and the medium was supplemented with the indicated cytokines (IL-1ß,
IL-6, IL-12, IL-15, IFN-, TNF-, and GM-CSF) at 50 ng/ml and
further incubated for 24 h. Cells without preincubation were
stimulated with the indicated cytokines at a concentration of 50 ng/ml
for 24 h. Then, cells were harvested and prepared for FACS
analysis or Western blot detection. Cell supernatants were collected,
highly centrifuged to pellet floating cells, and tested for the
presence of sFasL by sFasL ELISA as recommended by the manufacturer.
Cell coculture experiments
Coculture experiments were performed to determine the induction
of apoptosis in Jurkat T cells, which were cocultured with
cytokine-stimulated keratinocytes for 24 h. Keratinocytes were
plated at a density of 2–3 x 105/ml in six-well
tissue culture plates. Reaching confluence of 70–85% cells were
stimulated with IFN- (50 ng/ml) for 24 h with or without prior
preincubation with IL-10 (100 ng/ml) or TGF-ß1 (100 ng/ml). Jurkat T
cells were stimulated for 2 days with Con A (1 µg/ml) or were
cultured for 48 h with plate-bound anti-CD3 mAb 145-2C11 (1
µg/ml). Before coculture experiments, activated lymphoblasts (1
x 106) were washed and cocultured with nonstimulated or
cytokine-stimulated keratinocytes for 24 h in RPMI 1640 (10%
FCS). Thereafter, Jurkat T cells were harvested and analyzed for
apoptosis by flow cytometry. In FACS analysis keratinocytes
contaminating Jurkat T cell preparations were excluded by size.
Positive control experiments were performed using culture of Con
A-stimulated Jurkat T cells (1 x 106/ml) with
recombinant human soluble FasL (100 ng/ml), anti-Fas mAb (1 µg/ml,
clone CH-11), actinomycin D (1 µg/ml), and PMA (10 ng/ml)/Ca
ionophore A23187 (500 ng/ml), respectively. The Fas:Fc-IgG fusion
protein (20 µg/ml), known to block induction of apoptosis via Fas
receptor in Jurkat T cells, was used to control the specificity of
apoptosis. For a control, cell coculture was also performed in the
presence of unspecific mouse IgG. To strengthen further the specific
role of FasL expressed on keratinocytes for the induction of apoptosis
in cocultured Jurkat T cells, coculture experiments were performed with
COS-7 cells and L929 fibroblasts known to express no surface FasL Ag.
Flow cytometry
Analysis of Fas and FasL expression. Epidermal cells were harvested by incubation with trypsin/EDTA and washed twice with FACS buffer (PBS and 2% FCS). Cells (1 x 106) were incubated for 1 h at 4°C with anti-Fas mAb (F22120, Transduction Laboratories; 10 µg/ml), anti-FasL Ab (F37720, Transduction Laboratories; 10 µg/ml), or an isotype-matched control IgG1 Ab (Dianova). After washing the cells were stained with the second revealing FITC-conjugated goat anti-mouse IgG(H+L) F(ab')2 (1 µg/ml; Dianova) for 1 h at 4°C. Fluorescence staining of formaldehyde-fixed cells was analyzed using FACScan (Becton Dickinson, Heidelberg, Germany). The data are presented as mean fluorescence intensity (MFI) after subtraction of background staining (IgG control) or as the percentage of Fas/FasL-expressing or apoptotic cells, reflecting the percentage of cells with fluorescence above the control level (0.5%). Data were analyzed using CELL-LYSIS II software (Becton Dickinson).
Analysis of intracellular FasL. For intracellular staining of FasL, cells were permeabilized with a permeabilization solution (catalogue no. 340457) from Becton Dickinson (Erembodegem-Aalst, Belgium) according to the instructions provided. Staining of cell surface FasL was ruled out by prior protein digestion of cell surface molecules by papain for 20 min (1 mg/ml papain, 1 mM EDTA, 20 mM cysteine, and 80 mM Na2HPO4). Subsequently, cells were washed with PBS (2% (v/v) FCS) and permeabilized for 10 min in the dark. After staining with anti-FasL mAb (F37720) and FITC-labeled F(ab')2 of goat anti-mouse IgG, fluorescence was determined by flow cytometry.
Detection of apoptosis
TUNEL assay. To determine free 3'-OH groups of fragmented DNA in apoptotic Jurkat T cells the TUNEL assay was performed. The labeling was performed as recommended by the manufacturer (Boehringer Mannheim). The percentage of DNA fragmentation was determined by FACScan. For negative control staining the labeling enzyme was omitted. Data are presented as the percentage of cells showing fluorescence above the control level.
FITC-annexin V binding. The binding of FITC-annexin V was used to examine the exposure of phosphatidylserine on early apoptotic primary keratinocytes (30). The staining was conducted according to the recommendation of the manufacturer. After staining, the cells were immediately analyzed by flow cytometry. Only cells that stained positively for annexin V (fluorescence channel 1) and were not stained by propidium iodide (fluorescence channel 2) are presented as the percentage of apoptotic cells with fluorescence above the control level (0.5%).
Western blot analysis
Preparation of cellular protein extracts. To obtain whole cell lysates, epidermal cells were harvested by trypsinization and resuspended in Tris-Cl (50 mM; pH 7.5) containing 150 mM NaCl, 1% (v/v) Nonidet P-40, 1 mM EGTA, 1 mM PMSF, 1 mM DTT, and 30 µg/ml aprotinin. Cell disruption was conducted by rapid freeze-thawing five times. The completeness of cell disruption was verified by toluidine staining. After centrifugation at 300 x g for 20 min, the supernatants were collected, and protein determination was performed by Bradford staining with BSA as the protein standard (31).
Immunoblotting analysis of FasL. For Western blot analysis up to 15 µl of protein extracts (20–30 µg) were denatured by boiling in equal amounts of reducing 2x SDS-PAGE sample buffer (0.1 M Tris-Cl (pH 6.8), 0.1 M DTT, 0.2% (w/v) bromophenol blue, 4% (w/v) SDS, and 20% (v/v) glycerol). The samples were separated by SDS-PAGE (10–12%) according to the method of Laemmli (32) and transferred to ECL-Hybond-nitrocellulose membranes (Amersham, Braunschweig, Germany) by semidry electroblotting. Subsequently, unspecific binding sites were blocked by treatment with PBS (5% (w/v) BSA and 0.1% (v/v) Tween-20) at room temperature for 2 h. The blots were probed with anti-FasL Ab (0.25 µg/ml; F37720, Transduction Laboratories) in PBS (1% BSA and 0.1% Tween-20) for 3 h followed by a sheep anti-mouse HRP-conjugated secondary Ab (0.4 µg/ml). After extensive washing steps the blots were visualized using enhanced chemiluminescence detection reagents and ECL-Hybond film (Amersham).
Statistics
If not stated otherwise, the results obtained by flow cytometry studies are presented as the mean and SEM (MFI ± SEM or percentage of cells ± SEM) from three independently performed experiments. The significance was evaluated by nonpaired two-sided Student’s t test for independent means.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine constitutive FasL surface expression on human
epidermal cells we analyzed cells of the human epithelial cell line
A549, the human keratinocyte cell line HaCaT, freshly isolated
epidermal cells, and cultured primary human keratinocytes. The cells
were stained with anti-FasL mAb and analyzed by flow cytometry. As
shown in Fig. 1
, A and
B, the transformed epithelial cell line A549 expressed the
highest level of cell surface FasL (MFI = 163 ± 34); 56
± 8% cells stained positively for FasL. In contrast, freshly isolated
human epidermal cells stained negatively for FasL, but primary
keratinocytes cultured in vitro to subconfluence expressed FasL
constitutively on their cell surface (MFI = 130 ± 26).
Although only a subset of these primary keratinocytes (13 ± 2%;
Fig. 1B) expressed FasL in a constitutive manner, primary
keratinocytes expressed a higher FasL amount per cell compared with
A549 cells. A constitutive FasL expression was also observed on cells
of the human keratinocyte cell line HaCaT. Similar to primary
keratinocytes only a subset of about 10 ± 3% stained positively
for FasL (Fig. 1B). However, the FasL expression per cell
was less pronounced. A similar expression pattern was obtained after
staining with another anti-FasL mAb (clone H11, Alexis Corp.; data not
shown). For a control, the fibroblast cell line L929 and COS-7 cells,
known to be FasL-, stained negatively for FasL.
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C shows
a representative Western blot prepared from whole cell lysates. In
analogy to the results obtained by FACS analysis whole cell lysates
from A549 cells, primary keratinocytes, and HaCaT cells stained
positively for FasL (Fig. 1C, lanes 1,2, and 3, respectively), and HaCaT cells expressed only
a small amount of FasL. A whole cell lysate of Jurkat T cells activated
with Con A for 2 days served as a positive control (lane
4). The expected lane in the range of 37 kDa was only visible
after staining with anti-FasL mAb. Addition of isotype-matched
control Ab did not led to any positive staining of the blot (not
shown).
Enhanced expression of cell surface FasL on keratinocytes
stimulated with IFN-, TNF-, IL-1ß, and IL-15
Next, we examined whether cytokines might influence FasL
expression on primary keratinocytes. The cells were stimulated with 50
ng/ml of each cytokine for 24 h and thereafter analyzed for their
FasL expression by FACS analysis. Fig. 2A shows the results of these
experiments. We observed a significant 1.5- to 2.5-fold increase in
FasL expression on the cell surface of keratinocytes stimulated with
IFN-, TNF-, IL-1ß, and IL-15. In contrast, stimulation of the
cells with IL-6, IL-12, GM-CSF, IL-10, and TGF-ß1, respectively, did
not modulate the constitutive FasL expression pattern (data not shown).
As depicted in Fig. 2B, the stimulatory effect was dose
dependent. At a concentration of 50 ng/ml, IFN- and IL-1ß induced
a maximal up-regulation of FasL on primary keratinocytes. In contrast,
keratinocytes stimulated with TNF- up to 200 ng/ml showed a
permanent increase in FasL expression. Kinetic studies revealed that
FasL expression was up-regulated up to 48 h after cytokine
stimulation, reaching its maximum as early as 24 h after cytokine
priming (not shown).
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-stimulated keratinocytes might be due to a transport
mechanism of intracellular prestored FasL molecules to the plasma
membrane or to a new synthesis of FasL molecules, we stimulated primary
keratinocytes with IFN- (50 ng/ml) in the presence of nontoxic
concentrations of the protein synthesis inhibitor cycloheximide for
24 h. Cycloheximide completely inhibited the IFN--mediated
up-regulation of cell surface FasL expression (data not shown). In
analogy to cell surface-bound FasL, the increase in intracellular FasL
due to stimulation with IFN- was totally abolished in the presence
of cycloheximide. Immunoblot studies performed with whole cell lysates
confirmed these results (data not shown). These data indicate that the
observed IFN--mediated up-regulation of intracellular as well as
cell surface-bound FasL is dependent on new protein synthesis. Similar
data were obtained concerning FasL expression due to activation with
IL-1ß and TNF-, respectively.
Next, we wondered whether FasL might be secreted into the culture
supernatant following cytokine stimulation. Cell supernatants of
primary keratinocytes stimulated with IFN- (50 ng/ml) or TNF- (50
ng/ml) for 6–48 h were analyzed for sFasL by sFasL ELISA. Remarkably,
no sFasL was detectable in the cell supernatants of human primary
keratinocytes. In contrast, human PBMCs (1 x 106/ml)
activated with Con A (1 µg/ml) secreted up to 600 pg/ml FasL within
24 h of incubation (data not shown).
Monolayers of keratinocytes activated with IFN- show an
increased apoptosis rate
Recently, it was reported that stimulation of keratinocytes with
IFN- increases the expression of Fas. Since Fas as well as FasL
expression increased simultaneously on IFN--stimulated cells, the
question arises whether apoptosis might be induced in an autocrine
fashion. Therefore, we stimulated monolayers with different doses of
IFN- and cultured them for 48 h. After 24-h culture no
significant increase in dead cells compared with the medium control was
found in the cell supernatants. On the contrary, as shown in Fig. 3A, after a culture time of
48 h we observed a small, but significant, dose-dependent increase
in dead cells within the cell supernatants. Compared with the control
(4% of dead cells) IFN--stimulated (250 ng/ml) monolayers showed a
3-fold increase in dead detached cells (12%;
y1-axis). Furthermore, the cell number of viable
cells within the monolayer decreased significantly up to 30% in a
IFN- dose-dependent manner (y2-axis). To
determine whether these apoptotic/cytotoxic events are mediated by
autocrine Fas/FasL interaction we determined apoptotic cells still
within the monolayer by staining with FITC-annexin V. Fig. 3B shows a representative result from our experiments.
Nearly 50–60% of the cells stimulated with IFN- (100 ng/ml)
stained positively for phosphatidylserine (MFI =
>102) after 48 h of culture. This percentage of
apoptotic cells was also obtained by staining with propidium iodide
(data not shown). That Fas/FasL-induced apoptosis was mainly
responsible for the observed FITC-annexin V staining was verified by
blocking experiments using Fas:Fc-IgG fusion protein (20 µg/ml). It
should be mentioned that despite such a high number of apoptotic
keratinocytes within the monolayers they were still confluent.
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induced apoptosis in cocultured Fas+ T cells
Cells of immune-privileged sites are protected from cytotoxic
effector cells by their high constitutive FasL expression. Therefore,
we wondered whether an enhanced FasL expression on IFN--activated
keratinocytes might induce apoptosis in cocultured Fas+
Jurkat T cells. For this purpose, we cocultured Fas+ Jurkat
T cells with nonstimulated and IFN--stimulated primary human
keratinocytes for 24 h and determined apoptotic T cells by TUNEL
staining. Fig. 4A reveals the
frequency of apoptotic Fas+ Con A-activated Jurkat T cells
cocultured with confluent keratinocyte monolayers (gray bars). As
shown, prior activation of primary keratinocytes with IFN-
significantly increased the apoptosis of cocultured Jurkat T cells
compared with that in cocultures performed with nonstimulated
keratinocytes (from 14 ± 4 to 45 ± 8%). By addition of
Fas:Fc-IgG, which is known to inhibit the Fas/FasL interaction, this
proapoptotic effect was nearly abolished (20 ± 6%), whereas
control IgG was ineffective (Fig. 4A). Furthermore, COS-7
cells and L929 fibroblasts, which stained negatively for cell surface
FasL, induced no apoptosis in cocultured Jurkat T cells (data not
shown). For positive control staining, the percentages of apoptotic
Jurkat T cells incubated with sFasL, an agonistic anti-Fas IgM,
actinomycin D, or activated with PMA plus calcium ionophore A23187 were
determined (black bars). It is noteworthy that Con A-activated Jurkat T
cells cocultured with nonstimulated keratinocytes showed a lower
percentage of apoptosis than cells cultured with medium only in
uncoated wells.
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B shows
the results from two representative experiments. As demonstrated, T
cells previously activated with Con A (Fig. 4B.1) and
anti-CD3 Ab (Fig. 4B.2), respectively, and cocultured with
IFN--activated keratinocytes showed similar percentages of
TUNEL-positive cells. Again, in both models induction of apoptosis was
greatly inhibited in the presence of Fas:Fc-IgG, verifying that
lectin-mediated cytotoxicity can be excluded from our in vitro
coculture system.
Anti-inflammatory cytokines (IL-10, TGF-ß1) counter-regulate
IFN--mediated up-regulation of FasL on keratinocytes
The cytokines IL-10 and TGF-ß1 are well known for their
anti-inflammatory capacity. To analyze whether both IL-10 and
TGF-ß1 are able to modulate increased FasL expression on
IFN--stimulated keratinocytes, we preincubated primary keratinocytes
for 24 h with either of these cytokines and stimulated them
further for 24 h in the presence of IFN-. Preincubation of
keratinocytes with IL-10 (100 ng/ml) inhibited IFN--induced
up-regulation of FasL (Fig. 5,
A and B). Furthermore, pretreatment of
IFN--stimulated cells with TGF-ß1 diminished FasL expression about
20–25% (Fig. 5B). Neither IL-10 nor TGF-ß1 alone
modulated the constitutive FasL expression on cultured keratinocytes
(data not shown).
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, apoptosis in cocultured T cell was
diminished up to 90% by prior treatment of keratinocytes with IL-10
(Fig. 6A) as well as TGF-ß1 (Fig. 6B). These
unexpected high inhibition rates suggest that FasL molecules might be
expressed on keratinocytes up to a critical density to induce apoptosis
in cocultured T cells in an effective way.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our data clearly demonstrate that the expression of FasL on primary
human keratinocytes is up-regulated by several proinflammatory
cytokines (IL-1ß, TNF-, IFN-, and IL-15) and is
counter-regulated by anti-inflammatory cytokines (IL-10 and
TGF-ß1). By stimulation with IL-1ß, TNF-, IFN-, and IL-15,
respectively, both the number of FasL-positive cells and the expression
level of FasL increased significantly (Fig. 2). Cycloheximide
completely inhibited cytokine-mediated up-regulation of FasL,
demonstrating dependence on new protein synthesis. The observation that
up-regulation of FasL on keratinocytes is promoted by a variety of
cytokines in a redundant manner supports the idea of an important
physiological process.
Since epidermal keratinocytes are a well established source at least of
some of these cytokines (3, 25, 26, 27, 37), cell surface expression of
FasL expression on their cell surface might be enhanced during
inflammatory skin processes in an autocrine fashion. Moreover,
activated cytokine-expressing T cells infiltrating the inflammatory
skin may also contribute to this process in a paracrine fashion, e.g.,
by secreting IFN-.
Fas/FasL interaction leading to apoptotic epidermal cell death was found during normal epithelial cell turnover (38), in graft vs host disease (7), in autoimmune disorders (8, 9), and upon detrimental UV irradiation (10). Whether under these circumstances cytokines might be involved in the increased apoptosis pattern by up-regulation of FasL remains to be determined.
We measured a very high constitutive Fas and FasL expression on
epithelial cells of the transformed cell line A549 (37 and 57%,
respectively). However, like monocyte-derived macrophages, which were
also found to express Fas and FasL constitutively (39), they did not
undergo spontaneous apoptosis. In addition, we were not able to induce
apoptosis in Fas+ primary keratinocytes solely by the
addition of agonistic Abs or sFasL (data not shown). Therefore, Fas and
FasL molecules constitutively expressed or the signal transduction
pathway downward to the Fas receptor protein seem to be functionally
inactive in nonstimulated epidermal cells. In contrast, activation of
keratinocytes with IFN- increased cell surface expression of
functional Fas, leading to an increased apoptosis rate following
cross-linking of this receptor even with soluble Fas-targeting
molecules (21, 22, 40). Thus, both Fas and FasL are up-regulated by
IFN-, and this explains our observation on the pronounced apoptosis
rate in IFN--treated keratinocyte cultures (Fig. 3B).
These in vitro results correspond to villus epithelial damage in vivo
in IFN--stimulated mutant mice lacking intraepithelial lymphocytes
(38). In addition, IFN- alone and in combination with activation of
the Fas pathway induces apoptosis in A549 cells by an IFN--induced
expression of an effector of apoptosis, the cysteine protease
IL-1ß-converting enzyme (41). These results indicate that IFN-
supplies proapoptotic activity in epidermal cells by different
mechanisms. The physiological significance of this process may be the
limitation of growth factor-induced epidermal proliferation in
inflammatory skin. This view is supported by the recent observation
that hyperplastic keratinocytes from untreated psoriatic plaques did
not express FasL (23).
In contrast to activated monocytes and T cells, no soluble FasL was measured in the cell supernatants of cytokine-activated keratinocytes, suggesting that sFasL molecules are not involved in the apoptosis induction of Fas+ T cells. The apoptotic potential of soluble FasL is contradictory (40, 42, 43, 44, 45). One may speculate whether in vivo keratinocytes only induce apoptosis in skin-infiltrating T cells via direct cell-cell contact. Within the inflamed skin a local induction of apoptosis would be the consequence. On the contrary, our data concerning the release of sFasL from activated PBMCs led us to assume that activated T cells can mediate apoptosis of activated keratinocytes via both the membrane-anchored (cell-cell contact) and the soluble FasL (paracrine action) pathway.
Primary keratinocytes stimulated with IFN- induced apoptosis in
cocultured Fas+ Jurkat T cells. This apoptotic effect was
almost completely inhibited by addition of Fas:Fc-IgG, verifying that
apoptosis was due to Fas/FasL interaction. However, the enhancement of
apoptosis in cocultured Fas+ Jurkat T cells due to
pretreatment of keratinocytes with IFN- was much more impressive
than the IFN--mediated up-regulation of cell surface-bound FasL on
keratinocytes. Therefore, one may hypothesize whether IFN- might
support FasL-mediated killing by enhanced expression of molecules other
than FasL. In this regard, Sayama et al. observed a pronounced ICAM-1
expression on IFN--stimulated keratinocytes (21). The interaction of
ICAM-1 and LFA-1 supporting cell-cell adhesion is considered to be
important for cellular cytotoxicity (46). Furthermore, cytokines
released by the cocultured cells themselves might cooperate with
apoptotic signaling pathways, i.e., the Fas pathway. IFN--induced
expression of the proapoptotic cysteine protease IL-1ß-converting
enzyme in A549 cells was recently reported (41). These data suggest
that Fas/FasL-dependent killing of T cells may be additionally
potentiated by mechanisms other than enhanced cell surface expression
of FasL.
It remains to be determined whether enhanced FasL expression on cytokine-activated keratinocytes may limit an inflammatory process in the skin in vivo. A similar strategy, i.e., induction of apoptosis by Fas/FasL interaction in invading lymphocytes is an important mechanism for immune privilege (19). In this regard, allogeneic islets cotransplanted with Sertoli cells expressing a high amount of FasL or with syngeneic myoblasts transfected with FasL resulted in prolonged islet allograft survival (47, 48). One may hypothesize whether allogeneic skin transplants genetically engineered to express an enhanced level of cell surface-bound FasL might contribute to prolonged skin graft survival.
Some evidence accumulated that epidermal cells are able to synthesize
and release IL-10 and TGF-ß1 (49, 50). Since IL-10 and TGF-ß1 are
well known for their anti-inflammatory and immune-modulatory
capacity (51, 52, 53, 54), we were interested to analyze FasL expression on
IFN--stimulated keratinocytes after preincubation with IL-10 and
TGF-ß1, respectively. Neither IL-10 nor TGF-ß1 alone modulated
constitutive FasL expression, but they significantly diminished the
up-regulation of FasL on IFN--activated keratinocytes (Fig. 5).
Preliminary results also reveal inhibitory effects on IL-1ß-mediated
FasL up-regulation (data not shown). This down-regulation of FasL
nearly abolished apoptosis in cocultured Fas+ T cells (Fig. 6). Therefore, the amount of FasL expressed on the cell surface on
keratinocytes coincided with the ability to induce apoptosis in
cocultured T cells. One may assume that the pronounced up-regulation of
functionally active FasL by proinflammatory cytokines and
counter-regulation by IL-10 and TGF-ß1 play a central regulative role
for keratinocytes concerning cell turnover during inflammatory skin
processes. A reduced expression of FasL on keratinocytes might protect
the latter from physiological apoptosis, a scenario imaginable for
chronic inflammatory processes, e.g., psoriasis (23). Interestingly,
there was also an inhibitory effect of TGF-ß1 on FasL expression in
human activated T cells recently reported (55). It is at least one
mechanism by which TGF-ß1 blocks activation-induced cell death,
allowing expansion of effector cells and the generation of memory T
cells.
In contrast, cytokine-mediated up-regulation of FasL expression on keratinocytes might be important for tissue homeostasis as well as for induction of apoptosis in skin-infiltrating T cells and granulocytes (56, 57). Therefore, enhanced FasL expression might be responsible for both prevention of overwhelming reactions (including hyperplasia of keratinocytes) and limitation of cutaneous inflammation. Any disturbances in this process might have pathophysiological consequences. Moreover, it may be speculated that insufficient FasL on keratinocytes or of functionally active Fas on skin-infiltrating T cells could be involved in the pathogenesis of cutaneous T cell lymphoma.
In summary, our presented data supply further information for understanding the keratinocyte/T cell cross-talk during inflammatory skin reactions. Complex cytokine networks consisting of pro- and anti-inflammatory cytokines established during inflammatory skin diseases may determine the degree of FasL-induced apoptosis in keratinocytes themselves as well as in skin-infiltrating effector cells.
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Footnotes |
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2 Abbreviations used in this paper: FasL, Fas ligand; sFasL, soluble FasL; MFI, mean fluorescence intensity.
Received for publication August 20, 1998. Accepted for publication April 6, 1999.
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References |
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-inducing factor) messenger RNA and functional protein by murine keratinocytes. J. Immunol. 159:298.[Abstract]
-treated cultured keratinocytes. Arch. Dermatol. Res. 287:315.[Medline]
: evidence for synthesis and release upon stimulation with endotoxin or ultraviolet light. J. Exp. Med. 172:1609., TNF) in the induction of epithelial cell death and renewal. Eur. J. Immunol. 28:730.[Medline]
and Fas. Am. J. Physiol. 273:L921. and is mediated by intercellular adhesion molecule-1 (ICAM-1). J. Exp. Med. 167:1323.This article has been cited by other articles:
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  W. Yang, H. Li, P. W. Chen, H. Alizadeh, Y. He, R. N. Hogan, and J. Y. Niederkorn PD-L1 Expression on Human Ocular Cells and Its Possible Role in Regulating Immune-Mediated Ocular Inflammation Invest. Ophthalmol. Vis. Sci., January 1, 2009; 50(1): 273 - 280. [Abstract] [Full Text] [PDF]
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 R. Golan-Gerstl, S. B. Wallach-Dayan, G. Amir, and R. Breuer Epithelial Cell Apoptosis by Fas Ligand-Positive Myofibroblasts in Lung Fibrosis Am. J. Respir. Cell Mol. Biol., March 1, 2007; 36(3): 270 - 275. [Abstract] [Full Text] [PDF]
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 P.-L. Yao, Y.-C. Lin, P. Sawhney, and J. H. Richburg Transcriptional Regulation of FasL Expression and Participation of sTNF-{alpha} in Response to Sertoli Cell Injury J. Biol. Chem., February 23, 2007; 282(8): 5420 - 5431. [Abstract] [Full Text] [PDF]
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 M. H. Goldschmidt, J. S. Kennedy, D. R. Kennedy, H. Yuan, D. E. Holt, M. L. Casal, A. M. Traas, E. A. Mauldin, P. F. Moore, P. S. Henthorn, et al. Severe Papillomavirus Infection Progressing to Metastatic Squamous Cell Carcinoma in Bone Marrow-Transplanted X-Linked SCID Dogs. J. Virol., July 1, 2006; 80(13): 6621 - 6628. [Abstract] [Full Text] [PDF]
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 R. Arnold and W. Konig Respiratory Syncytial Virus Infection of Human Lung Endothelial Cells Enhances Selectively Intercellular Adhesion Molecule-1 Expression J. Immunol., June 1, 2005; 174(11): 7359 - 7367. [Abstract] [Full Text] [PDF]
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 L. Eidsmo, S. Nylen, A. Khamesipour, M.-A. Hedblad, F. Chiodi, and H. Akuffo The Contribution of the Fas/FasL Apoptotic Pathway in Ulcer Formation during Leishmania major-Induced Cutaneous Leishmaniasis Am. J. Pathol., April 1, 2005; 166(4): 1099 - 1108. [Abstract] [Full Text] [PDF]
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R. Barreiro, G. Luker, J. Herndon, and T. A. Ferguson Termination of Antigen-Specific Immunity by CD95 Ligand (Fas Ligand) and IL-10 J. Immunol., August 1, 2004; 173(3): 1519 - 1525. [Abstract] [Full Text] [PDF]
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K. A. Rezai, L. Farrokh-Siar, E. M. Gasyna, J. T. Ernest, and G. A. van Seventer The Effect of Type I and II Interferons on Human Fetal Retinal Pigment Epithelium-Induced Apoptosis in Jurkat T Cells Invest. Ophthalmol. Vis. Sci., July 1, 2003; 44(7): 3130 - 3134. [Abstract] [Full Text] [PDF]
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I. Viard-Leveugle, R. R. Bullani, P. Meda, O. Micheau, A. Limat, J.-H. Saurat, J. Tschopp, and L. E. French Intracellular Localization of Keratinocyte Fas Ligand Explains Lack of Cytolytic Activity under Physiological Conditions J. Biol. Chem., April 25, 2003; 278(18): 16183 - 16188. [Abstract] [Full Text] [PDF]
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 H. Yang, Y. Fan, and D. H. Teitelbaum Intraepithelial lymphocyte-derived interferon-gamma evokes enterocyte apoptosis with parenteral nutrition in mice Am J Physiol Gastrointest Liver Physiol, April 1, 2003; 284(4): G629 - G637. [Abstract] [Full Text] [PDF]
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Z. O-Quan Shi, M. J. Fischer, G. T. De Sanctis, M. R. Schuyler, and Y. Tesfaigzi IFN-{gamma}, But Not Fas, Mediates Reduction of Allergen-Induced Mucous Cell Metaplasia by Inducing Apoptosis J. Immunol., May 1, 2002; 168(9): 4764 - 4771. [Abstract] [Full Text] [PDF]
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K. R. Coulter, A. Doseff, P. Sweeney, Y. Wang, C. B. Marsh, M. D. Wewers, and D. L. Knoell Opposing Effect by Cytokines on Fas-Mediated Apoptosis in A549 Lung Epithelial Cells Am. J. Respir. Cell Mol. Biol., January 1, 2002; 26(1): 58 - 66. [Abstract] [Full Text] [PDF]
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Y. Wei, K. Chen, G. C. Sharp, H. Yagita, and H. Braley-Mullen Expression and Regulation of Fas and Fas Ligand on Thyrocytes and Infiltrating Cells During Induction and Resolution of Granulomatous Experimental Autoimmune Thyroiditis J. Immunol., December 1, 2001; 167(11): 6678 - 6686. [Abstract] [Full Text] [PDF]
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 G. Matute-Bello, W. C. Liles, C. W. Frevert, M. Nakamura, K. Ballman, C. Vathanaprida, P. A. Kiener, and T. R. Martin Recombinant human Fas ligand induces alveolar epithelial cell apoptosis and lung injury in rabbits Am J Physiol Lung Cell Mol Physiol, August 1, 2001; 281(2): L328 - L335. [Abstract] [Full Text] [PDF]
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J. E. Nichols, J. A. Niles, and N. J. Roberts Jr. Human Lymphocyte Apoptosis after Exposure to Influenza A Virus J. Virol., July 1, 2001; 75(13): 5921 - 5929. [Abstract] [Full Text] [PDF]
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M. Y. Braun, I. Grandjean, P. Feunou, L. Duban, R. Kiss, M. Goldman, and O. Lantz Acute Rejection in the Absence of Cognate Recognition of Allograft by T Cells J. Immunol., April 15, 2001; 166(8): 4879 - 4883. [Abstract] [Full Text] [PDF]
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G. J. Inman and M. J. Allday Apoptosis Induced by TGF-{beta}1 in Burkitt's Lymphoma Cells Is Caspase 8 Dependent But Is Death Receptor Independent J. Immunol., September 1, 2000; 165(5): 2500 - 2510. [Abstract] [Full Text] [PDF]
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A. Riccioli, D. Starace, A. D'Alessio, G. Starace, F. Padula, P. De Cesaris, A. Filippini, and E. Ziparo TNF-{alpha} and IFN-{gamma} Regulate Expression and Function of the Fas System in the Seminiferous Epithelium J. Immunol., July 15, 2000; 165(2): 743 - 749. [Abstract] [Full Text] [PDF]
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