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*
Division of Dermatology, Sunnybrook Health Science Centre, and
Amgen Institute, Department of Medical Biophysics and Immunology, University of Toronto, Toronto, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, has been shown to
induce apoptosis in a variety of cell types. Since UVB induces
keratinocytes to release TNF-, we hypothesized that TNF- is
involved in UVB-induced apoptosis in keratinocytes. In order to confirm
this hypothesis and to further delineate which type of TNF receptor
signaling mediates the apoptosis pathway, we performed both in vivo and
in vitro experiments using gene-targeted knockout mice lacking either
the TNF p55 receptor or the TNF p75 receptor. In the in vivo study,
wild-type and mutant mice were exposed to UVB, and apoptotic
keratinocytes were detected by examining DNA fragmentation using in
situ nick-end labeling. For the in vitro experiments, keratinocytes
derived from the wild-type and mutant mice were irradiated with UVB,
and the degree of apoptosis was determined by flow cytometry, nick-end
labeling of DNA, and a DNA ladder assay. Both in vivo and in vitro
studies demonstrated that the deletion of TNF receptor p55 could
suppress UVB-induced apoptosis in keratinocytes. Our observations
support the notion that TNF- is involved in UVB-induced keratinocyte
apoptosis, and demonstrate that p55 receptor signaling plays a pivotal
role in this event. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Environmental stimuli such as UV radiation, may trigger apoptosis
(5). Excess exposure of the skin of humans and laboratory
animals to UV light causes sunburn, photoaging, actinic keratosis, and
skin cancers (6). Ultraviolet B
(UVB)3 (290–320 nm) accounts for most of
the documented harmful biologic effects of sunlight. Epidermal cells
are considered to be a major target of UVB radiation since the vast
majority of UVB is absorbed within the epidermis, causing molecular
damage to nucleic acids (7). It is now clear that sunburn
cells caused by exposure of the skin to UVB, are keratinocytes that
have undergone apoptosis (5, 8). However, the molecular
mechanisms inducing keratinocyte apoptosis have not yet been fully
elucidated. Since TNF- can be induced by UVB (9) and
TNF- has been reported to induce apoptosis in various cell types
(10, 11), this cytokine has been thought to be involved in
UVB-induced keratinocyte apoptosis (8).
TNF- is a multifunctional cytokine produced by a variety of cell
types, including macrophages, T cells, mast cells, and keratinocytes
(12). Two distinct membrane receptors for TNF- have
been identified (13). The TNF- receptor, with a
molecular mass of 55 kDa, is referred to as the p55 receptor (Rp55 or
RI). The p75 receptor (Rp75 or RII) has a molecular mass of 75 kDa. The
two receptors are encoded by distinct genes; however, they are 
30%
homologous in their extracellular, cysteine-rich, and ligand-binding
regions. The genes for the p55 receptor and p75 receptor map to human
chromosomes 12 and 1, respectively, and in the mouse, to conserved
syntenic regions on chromosomes 6 and 4, respectively (14, 15). TNF-Rp55 is ubiquitously expressed, whereas TNF-Rp75 is
found predominantly on hemopoietic and endothelial cells
(16). TNF-Rp55 and TNF-Rp75 mediate distinct biological
activities. TNF-Rp55 signaling is thought to be involved in mediating
cytotoxicity, antiviral activity, fibroblast proliferation, and
induction of superoxide dismutase, while TNF-Rp75 signaling is involved
in the proliferation of thymocytes and cytotoxic T cells
(17). Although it is known that TNF- induces apoptosis
in a variety of cell types, the role of the two receptors in triggering
cell death is disputed. Some studies suggest that receptor p55 is the
main mediator of TNF--induced apoptosis (18), whereas
others contend that receptor p75 may be equally effective (11, 19).
In this study, we have used gene-targeted knockout (KO) mice deficient
in either TNF-Rp55 (20) or TNF-Rp75 (21) in
order to confirm the role of TNF- in UVB-induced keratinocyte
apoptosis, and to identify which TNF receptor mediates this apoptotic
pathway. The results of this study demonstrated that TNF p55 receptor
signaling plays a significant role in UVB-induced keratinocyte
apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The generation of TNF-Rp55 KO mice has been described previously (20) and this mutation was interbred 10 times into the C57BL/6 background. TNF-Rp75 KO mice on a C57BL/6 background were initially constructed by Dr. Moore (Genetech, South San Francisco, CA) (21). These mutant mice were maintained under a specific pathogen-free environment at the animal facility of Sunnybrook Health Science Centre, University of Toronto (Toronto, Canada). C57BL/6 mice were obtained from the Charles River Breeding Laboratories (Quebec, Canada) and used as a wild-type (WT) control. All mice were used at 8–12 wk of age. For keratinocyte cultures, newborn mice were used. Each experimental group consisted of five mice. All procedures were approved by the Animal Care Committee of Sunnybrook Health Science Centre.
In vivo UVB irradiation of mice
WT, TNF-Rp55, and TNF-Rp75 KO mice were shaved on their abdomens and exposed to UVB irradiation as described previously (22). Briefly, UVB irradiation was delivered with polychromatic light from a four-tube fluorescent sun lamp (FS20T12-UVB, National Biological, Twinsburg, OH). These lamps emit wavelengths mainly between 280 and 320 nm, peaking at 313 nm. The irradiation intensity was 0.45 mW/cm2 at a target distance of 15 cm, as measured by an IL-1400 A radiometer equipped with a SEL240/UVB 1/TD UVB detector with spectral sensitivity in the range of 280–320 nm (International Light, Montreal, Quebec, Canada). Following anesthetization by i.p. injection of pentobarbital (50 mg/kg), mice were given a single exposure of 200 mJ UVB light/cm2 on their abdomens. Negative control mice were treated in an identical way, but the UVB lamp was not switched on.
Nick-end labeling of DNA from apoptotic mouse skin cells
Twenty-four hours following UVB irradiation, skin biopsies were taken from the irradiated and control areas, fixed in buffered formaldehyde, and then embedded in paraffin. Five-micron paraffin sections were placed on slides pretreated with 0.01% aqueous solution of poly-L-lysine (300,000 m.w.; Sigma, St. Louis, MO). Slides were deparaffinized by heating overnight at 37°C and then rehydrated. The sections were analyzed by the terminal deoxynucleotidyltransferase (TdT)-mediated-deoxyuridine 5'-triphosphate (dUTP) nick-end labeling (TUNEL) technique using an in situ cell death detection kit (Boehringer Mannheim, Mannheim, Germany) (23). The TUNEL reaction preferentially labels DNA strand breaks generated during apoptosis (24). Labeling was performed according to the manufacturer’s instructions with minor modifications. The sections were preincubated with 20% normal bovine serum for 30 min at room temperature, and then 3'-OH termini of internucleosomal DNA strand breaks were labeled with fluorescein-dUTP, TdT, and alkaline phosphatase-labeled anti-fluorescein Ab. After the Fast red (Boehringer Mannheim) color reaction, counterstaining was performed with hematoxylin stain (Fisher Scientific, Fair Lawn, NJ). Sections were visualized by light microscopy. TUNEL results were quantified by counting 10 high power fields (hpf) of epidermis (magnification, x400) for each stained section. The number of apoptotic cells in the epidermis was expressed as a mean of 10 hpf.
Keratinocyte culture and in vitro UVB irradiation
Primary keratinocyte cultures were prepared from newborn WT and TNF receptor KO mice as described previously, but with some modifications (25). The skin samples were treated with 1% dispase II (Boehringer Mannheim) solution overnight at 4°C. Epidermal sheets were separated from the dermis and stirred in a trypsin-EDTA solution (0.05% trypsin and 0.53 mM EDTA) for 20 min at room temperature. The cell suspension was filtered through nylon mesh and centrifuged at 300 x g for 10 min. Cell pellets were resuspended in Eagle’s MEM with 10% heat-inactivated FBS (Life Technologies, Grand Island, NY) and plated at 2 x 106 cells/10-cm dish. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO2. The medium was removed and replaced with fresh medium every 2 days. For all experiments, third-passage cells were used at subconfluence.
For UVB irradiation, the medium was removed and keratinocytes were washed three times with prewarmed PBS and irradiated with UVB (0–20 mJ/cm2) in the presence of 0.5 ml PBS. Immediately after UVB treatment, the PBS was removed, replaced with medium, and the cells were cultured in Eagle’s MEM supplemented with 10% FBS for a further 24 h. The cells and supernatants were collected. Control cells were subjected to the identical procedure but were only sham irradiated.
Immunolabeling of TNF-Rp55 and TNF-Rp75
WT keratinocytes were labeled with anti-TNF-Rp55 or TNF-Rp75 Ab by a three-step immunostaining procedure (26). Briefly, 106 cells were incubated with rat anti-mouse TNF-Rp55 (100 µg/ml; Cedarlane, Hornby, Ontario, Canada), rat anti-mouse TNF-Rp75 (100 µg/ml; Cedarlane), or the equivalent amount of a rat IgG2a isotype control (Cedarlane) for 30 min at 4°C, washed, and then incubated with biotin-conjugated goat anti-rat IgG2a (Cedarlane) for 30 min on ice. After washing, cells were reacted with Streptavidin-phycoerythrin (Cedarlane) for 30 min on ice. Cells were then washed and analyzed using flow cytometry (FACScan, Becton Dickinson, San Jose, CA).
Apoptotic analysis by flow cytometry
Flow cytometric analysis was performed for quantification of cell death by apoptosis according to the method described previously (27). Due to DNA degradation and subsequent leakage from cells, apoptotic cells can be detected via their diminished staining with DNA-specific fluorochromes such as propidium iodide (PI) (Sigma). Keratinocytes (2 x 106 cells) from WT, TNF-Rp55 KO, and TNF-Rp75 KO mice were harvested and then fixed using 70% ethanol at 4°C for 1 h. The fixed cells were washed with PBS and incubated with 1 ml of hypotonic fluorochrome solution (50 µg/ml PI, 0.1% sodium citrate, 0.1% Triton X-100), and RNase-A (50 µg/ml) (Boehringer Mannheim) for 15 min at room temperature in the dark, and then kept on ice. DNA fluorescence was analyzed by quantitative flow cytometry using CellQuest Software. The percentage of apoptotic cells was identified by analyzing hypodiploid areas.
Nick-end labeling of DNA from apoptotic keratinocytes
Keratinocytes derived from WT, TNF-Rp55 KO, and TNF-Rp75 KO mice were exposed to UVB at a dose of 20 mJ/cm2, and 24 h later nick-end labeling of DNA in apoptotic cells was performed by using the APO-DIRECT kit (PharMingen, Mississauga, Ontario, Canada). A total of 2 x 106 cells were fixed with 1% paraformaldehyde solution, resuspended in 70% cold ethanol, and stored at -20°C. After rehydrating in PBS, the fixed cells were incubated in 50 µl of a solution containing FITC-labeled dUTP and TdT for 1 h at 37°C, and then resuspended in PI/RNase solution for 30 min at room temperature in the dark. Following incubation in staining solution, the cells were rinsed in PBS. Individual cells were analyzed by fluorescence microscopy using conjugated FITC (green) and PI (red) markers. The two negative control cells were treated in an identical way, however, one control was sham irradiated and the other control was incubated without TdT. FITC-labeled cells (apoptotic cells) were counted from about 100 randomly selected cells and expressed as a percentage of the total cells.
DNA fragmentation analysis
Detection of DNA fragmentation was performed after extraction of DNA, as previously reported, with some modifications (28). Untreated or irradiated keratinocytes (2 x 106) were collected, washed once with PBS, resuspended in 1 ml of lysis solution (10 mM Tris, pH 7.5, 1 mM EDTA, 0.2% Triton X-100) containing 5% SDS and proteinase K (0.5 mg/ml) (Sigma), and incubated overnight at 37°C. After centrifugation at 1000 x g for 20 min, the supernatants were extracted with phenol-chloroform. The genomic DNA was precipitated with ice-cold 100% ethanol. After centrifuging for 20 min at 1000 x g, the DNA pellet was resuspended in 15 µl of TE (10 mM Tris, pH 8.0, 1.0 mM EDTA), and then treated with 100 µg/ml RNase-A for 1 h at 37°C. Loading buffer (2 µl) was added to each sample. Samples were then electrophoresed on a 2% agarose gel containing 0.5 µg/ml ethidium bromide and visualized under UV light. The 100-bp ladder, which was used as a marker, was purchased from Pharmacia Biotech (Piscataway, NJ).
Reverse transcription and PCR
Total RNA was extracted from either mouse skin that had been
exposed to UVB or cultured keratinocytes, by the acid guanidinium
thiocyanate-phenol-chloroform method and RT-PCR was performed as
previously described (13, 29). Primer sets and positive
cDNA template controls for mouse TNF-, and ß-actin were obtained
from Clontech Laboratories (Palo Alto, CA). RNA from both TNF-Rp55 and
TNF-Rp75 expressing BW5147.3 cells (mouse T cell lymphoma-derived cell
line) was used as positive control. The sequences for each primer were
as follows: TNF-Rp55, 5'-GGA TAC AGT CTG CAG GGA GTG-3' and 5'-TCC ACC
GGG GAT ATC GGC ACA T-3'; TNF-Rp75, 5'-GAC AGG AAG GCT CAG ATG TGC T-3'
and 5'-GCA TTT CCG GGA ATA GCC AGG-3'; TNF-, 5'-ATG AGC ACA GAA AGC
ATG ATC CGC-3' and 5'-CCA AAG TAG ACC TGC CCG GAC TC-3'; and ß-actin,
5'-GTG GGC CGC TCT AGG CAC CAA-3' and 5'-CTC TTT GAT GTC ACG CAC GAT
TTC-3'. Specific cDNA obtained from reverse transcription was amplified
in a total volume of 10 µl containing 50 mM KCl, 10 mM Tris-HCl (pH
8.3), 1.5 mM MgCl2, 200 µM each of all four dNTPs,
10-6 M tetramethyl ammonium chloride, 10 pmol of each
primer, and 0.5 U of Taq DNA polymerase (Pharmacia Biotech).
The mixture was overlaid with 15 µl of mineral oil and PCR cycles
were performed in a Perkin-Elmer Cetus Thermal Cycler 480
(Perkin-Elmer/Cetus, Norwalk, CT) with denaturation at 94°C for 1
min, annealing at 55–60°C for 45 s, and extension at 72°C for
30 s. PCR signals for ß-actin, TNF-Rp55, TNF-Rp75, and TNF-
were obtained after 26, 32, 32, and 38 cycles, respectively. An aliquot
(4 µl) of the PCR product was electrophoresed on a 1.6% agarose gel
and visualized by ethidium bromide staining and UV illumination. After
photographing the gel, relative amounts of PCR products were determined
by scanning the negative films using a laser densitometer (LKB
2222-020, Ultroscan XL, Pharmacia). For relative quantity, the
densitometric value of each sample was normalized to that of
ß-actin.
Quantitation of TNF- protein
Keratinocytes were allowed to grow to subconfluence in Eagle’s
MEM with 10% FBS. The medium was replaced with PBS and the cells were
exposed to UVB (0–20 mJ/cm2). Immediately after UVB
treatment, the PBS was removed and replaced with the medium. After
another 24 h of incubation, the concentration of TNF- in
culture supernatants was measured using a commercially available ELISA
kit (Genzyme, Cambridge, MA) according to the manufacturer’s
procedures. TNF- concentration was determined from the linear
portion of a standard curve that was obtained using rTNF- (Genzyme).
The detection limit of the assay is 15 pg/ml. Each supernatant was
analyzed in triplicate.
Neutralization assay
Subconfluent keratinocytes from WT, TNF-Rp55 KO, and TNF-Rp75 KO
mice were exposed to UVB at a dose of 20 mJ/cm2 as
described above. Immediately after UVB irradiation, cells were
incubated with fresh medium containing various dilutions of polyclonal
rabbit anti-mouse TNF- (Genzyme) (0.5–10 µl/ml). We had
previously determined that 10 µl of anti-TNF- Ab can
neutralize up to 1000 U of rTNF- bioactivity. As a control, cultures
were incubated with medium containing identical concentrations of
rabbit IgG. After 24 h, the cells were collected and the
percentages of apoptotic keratinocytes were determined by flow
cytometry.
Statistical analysis
A minimum of three experiments was performed for each assay. All the data are expressed as mean values, with the SEM. The statistical significance was determined by either Student’s t test or ANOVA (when more than two groups were compared). A difference was considered to be statistically significant with p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In order to determine whether TNF--mediated keratinocyte
apoptosis involved signaling through TNF-Rp55 or TNF-Rp75, it was
necessary to determine that these receptors were present on murine
keratinocytes. Flow cytometric analysis demonstrated that both TNF-Rp55
and TNF-Rp75 were consistently detectable in WT murine keratinocytes
(Fig. 1
). Using primers specific for
mouse TNF-Rp55 or TNF-Rp75, PCR-amplified TNF-Rp55, and TNF-Rp75 cDNA
fragments were detected in WT murine keratinocytes, however, the latter
fragment was present at a lower level (Fig. 2).
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In order to determine if TNF- signaling involving either one of
the TNF receptors plays an important role in UVB-induced keratinocyte
apoptosis, we performed in vivo experiments using C57BL/6 mice that are
deficient in either TNF-Rp55 or TNF-Rp75. Mouse skin was irradiated
with UVB at a dose of 200 mJ/cm2 and, 24 h later,
biopsies were obtained, mounted on slides, and analyzed using the TUNEL
technique. As shown in Fig. 3,
TUNEL-labeled keratinocytes appeared in the epidermis of WT (Fig. 3a), TNF-Rp55 KO (Fig. 3b), and TNF-Rp75 KO (Fig. 3c) mice after UVB irradiation. Light microscopy revealed
shrunken cells, with irregularly-shaped and condensed nuclei, which
differentiated them from their normal neighbors. Apoptotic
keratinocytes were absent from the sham-irradiated epidermis of WT mice
(Fig. 3d). As an additional negative control, we used
UVB-exposed samples in which TdT was omitted. No TUNEL-positive
staining cells were seen in this control (data not shown). The number
of keratinocytes/hpf within epidermal sections from skin irradiated
with UVB (200 mJ/cm2) was 50.1 ± 3.1 (mean ±
SEM). The numbers of TUNEL-positive cells/hpf from WT mice, TNF-Rp55 KO
mice, and TNF-Rp75 KO mice were 15.0 ± 1.2, 9.0 ± 0.6, and
13.6 ± 1.4, respectively. These results are reported in Fig. 4 as the absolute number of positive
keratinocytes/hpf. No statistical difference was observed between WT
and TNF-Rp75 KO mice; however, the number of apoptotic keratinocytes
was significantly lower in TNF-Rp55 KO mice, compared with WT mice or
TNF-Rp75 KO mice.
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In the in vitro experiments, keratinocytes isolated from the WT,
TNF-Rp55 KO, and TNF-Rp75 KO mice were grown to subconfluence and then
irradiated with UVB. After 24 h, direct microscopic examination of
the culture dishes revealed a dose-dependent increase in the number of
detached cells possessing condensed nuclei. Apoptosis was quantitated
by flow cytometry. To test the sensitivity of this method,
keratinocytes from WT mice were exposed to UVB (0–20
mJ/cm2), and showed a dose-dependent increase of subdiploid
(apoptotic) cells with increasing UVB (Fig. 5). Although UVB irradiation induced
keratinocyte apoptosis in WT, TNF-Rp55-deficient, and
TNF-Rp75-deficient keratinocytes, a significantly lower number of
apoptotic cells were found in TNF-Rp55-deficient keratinocytes at all
UVB doses tested (Fig. 5).
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shows the results
of an experiment in which cultured keratinocytes from WT (Fig. 6a), TNF-Rp55 KO (Fig. 6b), and TNF-Rp75 KO (Fig. 6c) mice were exposed to UVB (20 mJ/cm2), and
apoptosis was analyzed 24 h later. No apoptotic keratinocytes were
observed in the sham-irradiated WT keratinocytes (Fig. 6d)
or in the absence of TdT (data not shown). The FITC-labeled
keratinocytes was counted under a fluorescent microscope and the number
of apoptotic cells/100 cells was evaluated in three independent
experiments. The number of FITC-labeled cells in WT,
TNF-Rp55-deficient, and TNF-Rp75-deficient keratinocytes was 28.9
± 3.4, 15.2 ± 2.1, and 28.3 ± 3.2, respectively (Fig. 7). The degree of apoptosis in
TNF-Rp55-deficient keratinocytes was significantly lower than that in
WT keratinocytes or TNF-Rp75-deficient keratinocytes (p <
0.05).
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To detect DNA fragmentation in UVB-irradiated keratinocytes, DNA was extracted and analyzed electrophoretically. Within 24 h of UVB irradiation, both WT keratinocytes and TNF-Rp75-deficient keratinocytes demonstrated the discontinuous eletrophoretic pattern of DNA degradation into nucleosomal fragments, while the internucleosomal DNA fragmentation was not evident in TNF-Rp55-deficient keratinocytes (data not shown). DNA ladders were completely absent in WT keratinocytes that had not been irradiated (data not shown).
UVB induces TNF- mRNA expression
in skin and TNF- protein release from keratinocytes
To examine whether the in vivo dose of UVB used in these studies
was sufficient to up-regulate endogenous TNF- production in the
skin, RNA was extracted from WT mouse skin exposed to 200
mJ/cm2 UVB. RT-PCR for TNF- and ß-actin was performed
at 0 and 24 h after UVB irradiation, and PCR products were
resolved on an agarose gel. Although low levels of TNF- mRNA were
detectable before exposure to UVB, TNF- mRNA markedly increased
24 h after UVB irradiation (Fig. 8A). Densitometric analysis
revealed that this increase was 6.3-fold (Fig. 8B).
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in the
skin, cultured keratinocytes from WT, TNF-Rp55 KO, and TNF-Rp75 KO mice
were irradiated with the same doses of UVB used for the in vitro
apoptosis assays, namely 0 to 20 mJ/cm2. After 24 h,
TNF- levels were measured from the culture supernatant by ELISA. As
shown in Fig. 8
C, in WT keratinocytes, TNF- protein was
below the detection level in the supernatant from the sham-irradiated
controls. As the UVB dose increased to 5 mJ/cm2, 10
mJ/cm2, and 20 mJ/cm2, the TNF-
concentration also increased (80.3 ± 9.5 pg/ml, 103.1 ±
11.2 pg/ml, and 180.2 ± 13.8 pg/ml, respectively). Similar
dose-dependent induction of TNF- was observed for both TNF-Rp55 KO
and TNF-Rp75 KO mice (data not shown).
Anti-TNF- Ab inhibits UVB-induced
apoptosis
To further confirm the role of TNF- signaling in UVB-induced
apoptosis, we treated the keratinocytes with a neutralizing Ab against
murine TNF- immediately after UVB irradiation (20
mJ/cm2). Apoptosis was assessed using flow cytometric
analysis 24 h later. Neutralizing TNF- Ab caused a
concentration-dependent suppression of apoptosis in WT- and
TNF-Rp75-deficient keratinocytes (data not shown). As illustrated in
Fig. 9, treatment with anti-TNF-
Ab (1 µl/ml) significantly decreased the frequency of UVB-induced
apoptosis in WT and TNF-Rp75 KO mice (apoptotic cell count of 25.1
± 2.3% and 24.7 ± 2.5%, respectively), as compared with
keratinocytes treated with control rabbit IgG (39.1 ± 2.2% and
37.2 ± 1.9% of apoptotic cells, respectively). A decrease of
approximately 33–36% in apoptosis occurred in both UVB-irradiated WT
and TNF-Rp75-deficient keratinocytes treated with anti-TNF- Ab
(compared with the control IgG-treated cells). TNF- Ab treatment of
both WT- and TNF-Rp75-deficient keratinocytes reduced apoptosis to
approximately the same level as that seen in TNF-Rp55-deficient
keratinocytes, suggesting that all TNF- signaling for UVB-induced
apoptosis was via the TNF-Rp55 receptor. In contrast, neutralizing
TNF- Abs had no effect on apoptosis in TNF-Rp55-deficient
keratinocytes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cytokines are associated with the regulation of apoptosis
(10, 11, 35, 36, 37, 38, 39). Some cytokines such as IL-6 and IL-10
protect cells from apoptosis (35, 36). However, certain
cytokines such as IFN- and IL-2 induce apoptosis (38, 39). TNF- is involved in apoptosis of a variety of cell types
such as T cells, fibroblasts, epithelial cells, macrophages, and
carcinoma cells (11, 40, 41, 42).
Keratinocytes are the major source of a variety of cytokines, including
IL-1, IL-6, IL-7, IL-10, IL-12, IL-15, IL-18, and TNF-, as well as
chemokines (9, 43, 44). The constitutive production of
most of these cytokines by keratinocytes is very low, but can increase
in the presence of various stimuli, including UVB. UVB irradiation of
keratinocytes induces almost all tested cytokines except for IL-7
(45). To determine whether the dose of UVB irradiation
used in our experiments up-regulates TNF- production in mouse skin,
we examined the levels of TNF- mRNA and protein after UVB
irradiation. Our results confirm that UVB significantly induces
keratinocytes to synthesize and release TNF-.
TNF- has been thought to be involved in UVB-induced keratinocyte
apoptosis. Studies from Schwarz et al. (8) have provided
evidence that TNF- plays a role in UVB-induced apoptosis of murine
keratinocytes. The present study demonstrates that anti-TNF- Abs
reduced UVB-induced apoptosis. These findings support the notion that
TNF- plays an important role in UVB-induced keratinocyte apoptosis
(8).
Of the two TNF receptors, p55 and p75, the former has been implicated as the main mediator of apoptosis in nonlymphoid cells such as 3T3 cells, neurons, murine hepatocytes, and human hepatoma cells (41, 46, 47). TNF-Rp55 and another member of the TNF receptor superfamily, Fas (APO-1/CD95), are homologous in a 70-amino acid region known as the "death domain." This domain is required for transducing the death signal through binding such mediators as TNF-Rp55-associated death domain protein (TRADD), MORT1/Fas-associating protein with death domain (FADD), and receptor-interacting protein (RIP), which may act as adapter proteins in both the TNF-Rp55 and Fas-activated signaling cascades (18). FADD and TRADD were found to bind differentially to Fas and TNF-Rp55, as well as to each other, and both bound effectively to RIP. The death domain of TRADD recruits FADD, which is responsible for downstream signal transduction by recruiting cytosolic aspartate-specific cysteine proteases 8 (caspase-8) (48). The C-terminal region of caspase-8 is related to the caspase family, specifically to caspase-3. The activated caspase members can cleave various substrates that lead to morphological changes known to accompany apoptosis (49). Recently, it has been demonstrated that TNF-Rp75 may also be involved in apoptosis of activated human synovial T cells, activated murine T cells, and TA1 cells (11, 40). The induction of apoptosis by TNF-Rp75 is strongly associated with the down-regulation of Bcl-XL mRNA and protein expression in activated T cells (50).
The present study demonstrates both TNF-Rp55 and TNF-Rp75 expression in
murine keratinocytes. We utilized gene-targeted mutant mice lacking
either TNF-Rp55 or TNF-Rp75, to elucidate the role of TNF- in
UVB-induced keratinocyte apoptosis as well as to further delineate
which type of TNF receptor signaling mediates the apoptotic pathway.
Both in vivo and in vitro studies demonstrated that there was a
significant reduction of UVB-induced keratinocyte apoptosis in TNF-Rp55
KO mice, compared with WT mice or TNF-Rp75 KO mice. These results
suggest that TNF-Rp55, but not TNF-Rp75, plays an important role in
UVB-induced keratinocyte apoptosis.
Although significantly down-regulated, UVB-induced apoptosis of
keratinocytes in TNF-Rp55 KO mice was not completely abolished.
Neutralizing TNF- Ab did not completely inhibit UVB-induced
apoptosis of keratinocytes, even at a concentration 100-fold greater
than that necessary for complete neutralization of autocrine TNF-
bioactivity in the supernatants. Moreover, we were not able to induce
apoptotic cell formation in WT keratinocytes by the addition of
increasing concentrations of rTNF- (data not shown). This result
confirms previous reports, which demonstrated that TNF- alone is
insufficient to induce keratinocyte apoptosis (8). RNA or
protein synthesis inhibitors are necessary in order to trigger
TNF-Rp55-mediated apoptosis (41, 51). Two major
TNF-induced responses have been described: apoptosis and activation of
(NF-
B). Activation of NF-B can inhibit TNF-Rp55-mediated
apoptosis (52). Blockage of the protective pathway
encourages the induction of cytotoxicity by triggering the apoptotic
process (51). These studies suggest that, although TNF-
is clearly involved in UVB-induced keratinocyte apoptosis via TNF-Rp55,
other molecules likely play a role as well. In fact, it has been
demonstrated that the cytotoxicity of TNF- can actually be enhanced
by other cytokines, such as IL-1 and IFN-
(53).
It has been shown that the suppressor gene, p53, has a direct effect on apoptosis by down-regulating Bcl-2 expression and up-regulating Bax expression (54). It is also involved in UVB-induced apoptosis in keratinocytes and, in fact, levels of p53 protein in human epidermis are significantly increased following UV irradiation (34, 55). In addition, Fas/Fas ligand, which was shown to be expressed on keratinocytes, may also play an important role (56). However, UV light is known to directly stimulate Fas receptor itself, thereby activating the Fas-dependent death pathway independently of Fas ligand (57). Clearly, the exact role of these multiple components of the keratinocyte survival machinery requires further studies.
Nevertheless, our observations support the notion that TNF- is
involved in UVB-induced keratinocyte apoptosis and demonstrate that p55
receptor signaling plays a pivotal role in this complex event.
Understanding the mechanisms that regulate apoptosis and identifying
individual control points of the apoptotic pathway in keratinocytes
will likely have an impact on future strategies for therapeutic
intervention in UVB-related skin diseases.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Daniel N. Sauder, Division of Dermatology, Sunnybrook Health Science Centre, University of Toronto, 2075 Bayview Avenue, Toronto, ON, Canada M4N 3M5. E-mail address:
3 Abbreviations used in this paper: UVB, ultraviolet B; KO, gene knockout; hpf, high power fields; TNF-Rp55, TNF receptor p55; KO, knockout; TNF-Rp75, TNF receptor p75; WT, wild type; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxyuridine 5'-triphosphate nick-end labeling; PI, propidium iodide. TdT, terminal deoxynucleotidyltransferase; TRADD, TNF-Rp55-associated death domain protein; FADD, Fas-associating protein with death domain.
Received for publication May 21, 1998. Accepted for publication October 20, 1998.
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References |
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B activation by ultraviolet light not dependent on a nuclear signal. Science 261:1442. in the formation of sunburn cells. J. Invest. Dermatol. 104:922.[Medline]
: evidence for synthesis and release upon stimulation with endotoxin or ultraviolet light. J. Exp. Med. 172:1609. mediates DNA fragmentation in HL-60 and U937 cells and differentiation in HL-60 cells. Blood 80:1339.
RI. J. Exp. Med. 175:1353. and ligation of Fas antigen. J. Immunol. 155:4147.[Abstract]
2. Eur. J. Immunol. 26:3119.[Medline]
B in preventing TNF--induced cell death. Science 274:782.-inducing factor) messenger RNA and functional protein by murine keratinocytes. J. Immunol. 159:298.[Abstract]
-induced IL-7 gene expression in murine keratinocytes by interfering with IFN regulatory factors. J. Immunol. 158:5393.[Abstract]
B activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice. Immunity 7:715.[Medline]
-induced apoptosis by NF-B. Science 274:787.This article has been cited by other articles:
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  M. Kneilling, R. Mailhammer, L. Hultner, T. Schonberger, K. Fuchs, M. Schaller, D. Bukala, S. Massberg, C. A. Sander, H. Braumuller, et al. Direct crosstalk between mast cell-TNF and TNFR1-expressing endothelia mediates local tissue inflammation Blood, August 20, 2009; 114(8): 1696 - 1706. [Abstract] [Full Text] [PDF]
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 J. Menke, M.-Y. Hsu, K. T. Byrne, J. A. Lucas, W. A. Rabacal, B. P. Croker, X.-H. Zong, E. R. Stanley, and V. R. Kelley Sunlight Triggers Cutaneous Lupus through a CSF-1-Dependent Mechanism in MRL-Faslpr Mice J. Immunol., November 15, 2008; 181(10): 7367 - 7379. [Abstract] [Full Text] [PDF]
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 J. Chen, X. Cheng, M. Merched-Sauvage, C. Caulin, D. R. Roop, and P. J. Koch An unexpected role for keratin 10 end domains in susceptibility to skin cancer J. Cell Sci., December 15, 2006; 119(24): 5067 - 5076. [Abstract] [Full Text] [PDF]
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C. Cataisson, A. J. Pearson, S. Torgerson, S. A. Nedospasov, and S. H. Yuspa Protein Kinase C{alpha}-Mediated Chemotaxis of Neutrophils Requires NF-{kappa}B Activity but Is Independent of TNF{alpha} Signaling in Mouse Skin In Vivo J. Immunol., February 1, 2005; 174(3): 1686 - 1692. [Abstract] [Full Text] [PDF]
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 E. J. Horn, A. Albor, Y. Liu, S. El-Hizawi, G. E. Vanderbeek, M. Babcock, G. T. Bowden, H. Hennings, G. Lozano, W. C. Weinberg, et al. RING protein Trim32 associated with skin carcinogenesis has anti-apoptotic and E3-ubiquitin ligase properties Carcinogenesis, February 1, 2004; 25(2): 157 - 167. [Abstract] [Full Text] [PDF]
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R. M. Clancy, C. B. Backer, X. Yin, R. P. Kapur, Y. Molad, and J. P. Buyon Cytokine Polymorphisms and Histologic Expression in Autopsy Studies: Contribution of TNF-{alpha} and TGF-{beta}1 to the Pathogenesis of Autoimmune-Associated Congenital Heart Block J. Immunol., September 15, 2003; 171(6): 3253 - 3261. [Abstract] [Full Text] [PDF]
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 Y.-Y. He, J.-L. Huang, D. C. Ramirez, and C. F. Chignell Role of Reduced Glutathione Efflux in Apoptosis of Immortalized Human Keratinocytes Induced by UVA J. Biol. Chem., February 28, 2003; 278(10): 8058 - 8064. [Abstract] [Full Text] [PDF]
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D. S. Rosenthal, A. Velena, F.-P. Chou, R. Schlegel, R. Ray, B. Benton, D. Anderson, W. J. Smith, and C. M. Simbulan-Rosenthal Expression of Dominant-negative Fas-associated Death Domain Blocks Human Keratinocyte Apoptosis and Vesication Induced by Sulfur Mustard J. Biol. Chem., February 28, 2003; 278(10): 8531 - 8540. [Abstract] [Full Text] [PDF]
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C. M. Simbulan-Rosenthal, A. Velena, T. Veldman, R. Schlegel, and D. S. Rosenthal HPV-16 E6/7 Immortalization Sensitizes Human Keratinocytes to Ultraviolet B by Altering the Pathway from Caspase-8 to Caspase-9-dependent Apoptosis J. Biol. Chem., June 28, 2002; 277(27): 24709 - 24716. [Abstract] [Full Text] [PDF]
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S. Nakagawa, C. W. Koomen, J. D. Bos, and M. B. M. Teunissen Differential Modulation of Human Epidermal Langerhans Cell Maturation by Ultraviolet B Radiation J. Immunol., November 15, 1999; 163(10): 5192 - 5200. [Abstract] [Full Text] [PDF]
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K. Amdjadi and B. M. Sefton Ultraviolet Light-induced Stimulation of the JNK Mitogen-activated Protein Kinase in the Absence of Src Family Tyrosine Kinase Activation J. Biol. Chem., July 14, 2000; 275(29): 22520 - 22525. [Abstract] [Full Text] [PDF]
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S.-F. Shih, Y.-H. Wu, C.-H. Hung, H.-Y. Yang, and J.-Y. Lin Abrin Triggers Cell Death by Inactivating a Thiol-specific Antioxidant Protein J. Biol. Chem., June 8, 2001; 276(24): 21870 - 21877. [Abstract] [Full Text] [PDF]
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