close

Вход

Забыли?

вход по аккаунту

?

The intracellular 52-kd RoSSA autoantigen in keratinocytes is up-regulated by tumor necrosis factor ╨Ю┬▒ via tumor necrosis factor receptor I.

код для вставкиСкачать
ARTHRITIS & RHEUMATISM
Vol. 52, No. 2, February 2005, pp 531–538
DOI 10.1002/art.20851
© 2005, American College of Rheumatology
The Intracellular 52-kd Ro/SSA Autoantigen in Keratinocytes
Is Up-Regulated by Tumor Necrosis Factor ␣
Via Tumor Necrosis Factor Receptor I
Velia Gerl,1 Björn Hostmann,1 Christa Johnen,2 Aderajew Waka,1 Markus Gerl,1
Frank Schumann,2 Rolf Klein,1 Andreas Radbruch,3 and Falk Hiepe1
Objective. Previous studies have shown that the
nuclear Ro/SSA autoantigens involved in photosensitive
cutaneous lupus manifestations are regulated by ultraviolet B (UVB) irradiation. UVB exposure triggers the
release of tumor necrosis factor ␣ (TNF␣) from keratinocytes in the epidermis and from mast cells in the
dermis. The present study aimed to characterize the
effect of TNF␣ on messenger RNA (mRNA) and protein
expression of the intracellular 52-kd Ro/SSA autoantigen in primary human keratinocytes and to elucidate
the TNF␣ receptor (TNFR) signaling pathways mediating this effect.
Methods. Expression of 52-kd Ro/SSA mRNA in
primary human keratinocytes was investigated by quantitative real-time polymerase chain reaction (LightCycler system) using GAPDH as the housekeeping gene.
Expression of 52-kd Ro/SSA protein was studied by flow
cytometry after staining intracellular protein with IgG
purified from an anti–52-kd Ro/SSA–positive serum.
TNFR function was assessed by culturing cells in the
presence and absence of neutralizing antibodies directed against the TNFR subunits TNFRI and TNFRII.
Results. TNF␣-induced up-regulation of 52-kd
Ro/SSA mRNA expression peaked at 4 hours, followed
by up-regulation of intracellular 52-kd Ro/SSA protein
expression at 24 hours, independently of apoptosis.
Between different donors, a high variability of both
constitutive expression levels and TNF␣ -induced
changes in 52-kd Ro/SSA mRNA and protein expression
was observed. The up-regulatory effect of TNF␣ on
52-kd Ro/SSA mRNA and protein expression was inhibited by anti-TNFRI antibodies but enhanced by antiTNFRII antibodies.
Conclusion. The finding that TNF␣ up-regulates
52-kd Ro/SSA expression in keratinocytes via TNFRI
suggests that it may play a role in the pathogenesis of
anti-Ro/SSA–associated cutaneous lupus erythematosus.
Autoantibodies directed against the small RNP
particle Ro/SSA are mainly associated with connective
tissue diseases, especially Sjögren’s syndrome, systemic
lupus erythematosus (SLE), subacute cutaneous lupus
erythematosus (SCLE), neonatal lupus syndromes, undifferentiated connective tissue disease, and myositis,
and are occasionally detected in other autoimmune diseases, such as systemic sclerosis, rheumatoid arthritis,
primary biliary cirrhosis, and autoimmune hepatitis (1–4).
Anti-Ro/SSA antibodies are often detected together with anti-La/SSB antibodies in sera and are
strongly associated with photosensitive cutaneous eruptions in SCLE (5,6), neonatal lupus (7), and other forms
of lupus. These autoantibodies are therefore thought to
play a key role in the pathogenesis of these skin manifestations, as suggested by the characteristic deposition
of immunoglobulins and complement at the epidermal
junction. Lee and coworkers experimentally reproduced
this pattern of cutaneous IgG deposition by infusing
purified anti-Ro/SSA autoantibodies into human skin–
grafted mice (8). However, since the targets of these
Supported by grants from the Deutsche Forschungsgemeinschaft (SFB 421, C4) and the Competence Network Rheumatology
(C3.5).
1
Velia Gerl, PhD, Björn Hostmann, MD, Aderajew Waka,
Markus Gerl, MD, Rolf Klein, PhD, Falk Hiepe, MD, PhD: Charité
Hospital, Center for University Medicine in Berlin, and German
Rheumatism Research Center, Berlin, Germany; 2Christa Johnen,
PhD, Frank Schumann: Charité Hospital, Center for University Medicine in Berlin, Berlin, Germany; 3Andreas Radbruch, PhD: German
Rheumatism Research Center, Berlin, Germany.
Address correspondence and reprint requests to Falk Hiepe,
MD, PhD, Charité Hospital, Center for University Medicine in Berlin,
Department of Medicine (Rheumatology and Clinical Immunology),
Schumannstrasse 20/21, D-10117 Berlin, Germany. E-mail: falk.hiepe@
charite.de.
Submitted for publication July 26, 2003; accepted in revised
form November 15, 2004.
531
532
GERL ET AL
autoantibodies (52- and 60-kd Ro/SSA autoantigens)
are located in intracellular compartments, including the
nucleus and the cytoplasm (for review, see ref. 9), these
intracellular autoantigens must be accessible to the
autoantibodies in order to be pathogenic.
Several studies revealed that Ro/SSA autoantigens are expressed on the surface of keratinocytes,
which are major sites of immunologic damage in photosensitive cutaneous lupus after exposure to stress factors
such as ultraviolet (UV) light (10–13), estradiol (14,15),
cytotoxic prostaglandins (16), viral infections (17), oxidative stress (18), heat shock (19), and phorbol 12myristate 13-acetate (19). Ro/SSA autoantigens are also
present in larger apoptotic blebs (20). Antibodydependent cell-mediated cytotoxicity (ADCC) is an important mechanism by which anti-Ro/SSA autoantibodies can induce keratinocyte cytotoxicity after UVB
exposure (21–23).
Previous studies have shown that tumor necrosis
factor ␣ (TNF␣) can enhance anti-Ro/SSA antibody
binding to the surface of keratinocytes (24). Interestingly, UVB irradiation triggers the release of TNF␣
from keratinocytes in the epidermis (25,26) and from
mast cells in the dermis (27). Therefore, TNF␣ is
thought to be a key mediator in the pathogenesis of both
anti-Ro/SSA–associated lupus and photosensitive cutaneous lupus. The objective of the present study was
therefore to elucidate the role of TNF␣ in messenger
RNA (mRNA) and protein expression of the intracellular 52-kd Ro/SSA autoantigen in primary human keratinocytes. To better understand the regulatory mechanisms underlying 52-kd Ro/SSA autoantigen expression,
we also investigated the impact of the TNF␣ receptors I
and II (TNFRI and TNFRII) on the up-regulatory effect
of TNF␣.
MATERIALS AND METHODS
Cell culture. Primary human keratinocytes were isolated from the skin of healthy donors and cultured at 1 ⫻ 104
cells/cm2 in 75-cm2 culture flasks containing serum-free keratinocyte growth medium (EpiLife; Tebu-Bio, Offenbach, Germany) supplemented with 60 ␮M CaCl2, EpiLife defined
growth supplement (Tebu-Bio), 100 units/ml penicillin, and
100 ␮g/ml streptomycin (Gibco, Paisley, UK) at 37°C in an
atmosphere of 5% CO2 in air (28).
Cell treatment and analysis of apoptosis. Primary
human keratinocytes grown to 80–90% confluence were
treated for 1, 2, 4, 8, and 24 hours with 4 ng/ml of human
recombinant TNF␣ (Sigma, St. Louis, MO) as described
previously (24). Cells were harvested at the specified sampling
times, including floating cells detached during the apoptotic
process. Intact cells were detached from the flask bottom using
Accutase (PAA Laboratories, Linz, Austria), then washed with
phosphate buffered saline (Gibco) and stained with annexin
V–fluorescein isothiocyanate (FITC) (Becton Dickinson, Heidelberg, Germany) and propidium iodide as recommended by
the manufacturer. A FACSCalibur flow cytometer (Becton
Dickinson) was used to identify apoptotic cells, which tested
positive for annexin V and either negative or positive for
propidium iodide. The apoptotic effect of TNF␣ was calculated as the difference between the percentage of apoptotic
cells in the TNF␣-treated cell population and the percentage
of apoptotic cells in the untreated controls. Since the maximum range of error in repeated independent experiments was
⬃10% (data not shown), all differences ⬎10% were defined as
a significant effect. All experiments were carried out in duplicate; mean ⫾ SEM values are indicated.
Quantitative real-time polymerase chain reaction
(PCR). To keep degraded ribonucleotides from contaminating
the samples, annexin V–FITC–positive apoptotic cells were
sorted out using a FACSVantage cell sorter (Becton Dickinson). Messenger RNA was isolated from the nonapoptotic cell
population using the Oligotex mRNA direct kit (Qiagen,
Hilden, Germany). Real-time PCR was performed with a
LightCycler (Roche Diagnostics, Mannheim, Germany). Messenger RNA for 52-kd Ro/SSA was amplified and quantitatively analyzed using a 1-step mRNA amplification kit for
hybridization probes (Roche Diagnostics).
PCR reactions were set up in samples with a final
volume of 17 ␮l, including 10 ␮g mRNA from samples or
standards, 0.41 ␮M primers, and 2 ␮M hybridization probes.
The amplification program for 52-kd Ro/SSA included reverse
transcription at 55°C for 15 minutes and initial denaturation at
95°C for 3 minutes, followed by 45 cycles of denaturation at
94°C for 30 seconds, primer annealing at 59°C for 13 seconds,
and extension at 72°C for 20 seconds. Fluorescence of the
hybridization probe was measured after the annealing step.
The specificity of amplification was determined by melting
curve analysis. The relative amount of 52-kd Ro/SSA transcripts was normalized to the number of human GAPDH
transcripts found in the same reaction tube. Cloned Ro/SSA
(29) and GAPDH complementary DNA were used as the
quantification standards.
Messenger RNA for 52-kd Ro/SSA was amplified
using the primer sequences 5⬘-AGCAAAAAAACTTCCTGG
TTG-3⬘ (forward) and 5⬘-GAGCTGTGGCACCTTCGATC3⬘ (reverse). GAPDH amplification was carried out using the
primer sequences 5⬘-GCAGGGGGGAGCCAAAAG-3⬘ (forward) and 5⬘-AGGCAGGGATGATGTTCTGGA-3⬘ (reverse). The transcripts were detected using fluorescent hybridization probes provided by the manufacturer (TIB-MOLBIOL,
Berlin, Germany). Each PCR assay was run in triplicate. The
52-kd Ro/SSA and GAPDH copy numbers were estimated
from the threshold amplification cycle numbers using the
software supplied with the LightCycler.
Intracellular staining of 52-kd Ro/SSA protein. In
order to avoid false results due to apoptotic protein degradation, intracellular protein expression was investigated in keratinocytes exhibiting high viability using only the intact cells
attached to the flask bottom. Cells were fixed and permeabilized using the Fix & Perm cell permeabilization kit (An-derGrub Bioresearch, Kaumberg, Austria). Specific IgG antibodies purified from the serum of patients with only anti–52-kd
UP-REGULATION OF 52-kd Ro/SSA BY TNF␣ VIA TNFRI
Ro/SSA antibody reactivity were used to detect intracellular
52-kd Ro/SSA expression. Sole reactivity to 52-kd Ro/SSA was
confirmed by immunoblotting using Molt-4 cell extract (30).
IgG binding was detected using a phycoerythrin-labeled antihuman IgG (mouse clone G18-145; PharMingen, San Diego,
CA) as a secondary antibody. The samples were incubated with
the antibodies for 30 minutes at room temperature. Specific
binding of the anti-Ro/SSA antibody was confirmed by blocking the samples with the Ro/SSA affinity-purified antigen (Immunovision, Springdale, AR). Stained cells were enumerated
using a FACSCalibur flow cytometer. Apoptotic keratinocytes
were excluded based on their light-scatter characteristics.
Relative changes in expression of 52-kd Ro/SSA antigen in
response to TNF␣ treatment were calculated as the ratio of
specific antigen staining of treated versus untreated cells, as
described elsewhere (31).
Studies of TNFRI and TNFRII function. The role of
the TNF␣ receptors TNFRI and TNFRII in the up-regulation
of 52-kd Ro/SSA autoantigen expression by TNF␣ in human
keratinocytes was investigated by pretreating the cells with 0.5
␮g/ml neutralizing monoclonal antibodies directed against
human soluble TNFRI (sTNFRI) (IgG1 clone 16803; R&D
Systems, Minneapolis, MN) and/or human sTNFRII (IgG2a
clone 22221.311; R&D Systems) for 30 minutes and by further
incubating the cells with these antibodies in combination with
TNF␣ for up to 24 hours. Intracellular 52-kd Ro/SSA protein
and mRNA expression were measured as described above.
Statistical analysis. Nonparametric Mann-Whitney
tests for comparison of two independent groups were run on
GraphPad Prism 3.02 for Windows (GraphPad Software, San
Diego, CA). Values were calculated as the mean ⫾ SEM. P
values less than 0.05 were defined as statistically significant.
RESULTS
TNF␣-induced up-regulation of 52-kd Ro/SSA
mRNA transcription. Studies of 52-kd Ro/SSA mRNA
expression in annexin V–negative (nonapoptotic) human
epidermal keratinocytes were carried out by quantitative
real-time PCR. Constitutive expression of Ro/SSA was
low (2.3 ⫻ 102 copies of 52-kd Ro/SSA mRNA). Cells
from 5 donors were treated with TNF␣ to determine the
effect of TNF␣ on 52-kd Ro/SSA mRNA expression
over time. Ro/SSA expression data were normalized to
the number of GAPDH mRNA transcripts. We observed a time-dependent increase in 52-kd Ro/SSA
mRNA expression, which peaked at 4 hours. The upregulatory effect of TNF␣ was strong in 2 of the 5
donors, weak in 2 donors, and absent in 1 donor. At the
24-hour sampling time, 52-kd Ro/SSA mRNA expression had decreased sharply and was nearly as low as that
in the unstimulated controls (Figure 1). Encouraged by
these findings, we investigated 52-kd Ro/SSA mRNA
expression in keratinocytes from another 4 donors.
Variable degrees of TNF␣-induced up-regulation of
52-kd Ro/SSA mRNA expression were observed at 4
533
Figure 1. Transcription of 52-kd Ro/SSA mRNA up-regulated by
tumor necrosis factor ␣ (TNF␣). TNF␣ induced a time-dependent
increase in 52-kd Ro/SSA mRNA expression, which peaked at 4 hours.
After longer periods of incubation, the expression rate decreased
rapidly, falling almost to the level measured in the unstimulated
controls. The ratio of 52-kd Ro/SSA mRNA to GAPDH was calculated
as the mean of triplicate measurements. The extent of mRNA regulation varied from donor to donor. Strong up-regulation was found in
2 donors (solid circles and open squares), weak up-regulation in
another 2 donors (open diamonds and diamonds with an X), and no
response in 1 donor (solid triangles). Untreated keratinocytes did not
reveal any effects (results not shown).
hours. The up-regulatory effect of TNF␣ was strong in 3
of the 9 donors studied (Ro/SSA to GAPDH ratios of
12.8, 24.5, and 38.5), weak in 5 donors (ratios of 3.67,
3.76, 3.05, 4.86, and 2.65), and absent in 1 donor (ratio of
0.95).
TNF␣-induced up-regulation of intracellular
52-kd Ro/SSA protein expression. To determine
whether up-regulation of 52-kd Ro/SSA mRNA leads to
up-regulation of 52-kd Ro/SSA protein, we analyzed
samples from 6 additional donors by flow cytometry
using anti–52-kd Ro/SSA IgG. As expected, these tests
showed that constitutive intracellular expression of
52-kd Ro/SSA protein occurs in primary human keratin-
534
GERL ET AL
Figure 2. Slight up-regulation of intracellular 52-kd Ro/SSA autoantigen expression in primary human keratinocytes by tumor necrosis
factor ␣ (TNF␣). The cells were cultured with or without TNF␣ for up
to 24 hours, fixed, permeabilized, and stained for autoantigen expression. The stained cells were evaluated by fluorescence-activated cell
sorting analysis. The living cell fraction was gated according to
light-scatter characteristics (results not shown). The staining results
for a representative sample (donor 1) are presented as a fluorescence
histogram showing the 52-kd Ro/SSA fluorescence of untreated cells
(thick black line) and TNF␣-treated cells (thick gray line). 52-kd
Ro/SSA stained in the presence of the blocking autoantigen (thin gray
line) and fluorescence-conjugated secondary antibodies alone (antihuman IgG–phycoerythrin; thin black line) were used as controls.
Mean fluorescence intensities are given.
ocytes. Positive staining was neutralized by the affinitypurified Ro/SSA antigen and was therefore judged to be
specific. TNF␣ slightly up-regulated 52-kd Ro/SSA protein expression in 4 of 6 donors (Figure 2 and Table 1).
Up-regulation of antigen expression was identified as a
fluorescence shift in the whole cell population. The
baseline level and extent of up-regulation of 52-kd
Ro/SSA expression naturally varied from donor to donor. Western blot analyses performed using keratinocyte
protein extracts from 2 donors also revealed weak
up-regulation of 52-kd Ro/SSA protein expression (data
Table 1. Expression of 52-kd Ro/SSA protein in primary human
keratinocytes from different donors*
Donor
Untreated cells
TNF␣-treated cells
RCE
1
2
3
4
5
6
6.81 ⫾ 0.3
8.25 ⫾ 0.4
14.74 ⫾ 0.1
11.92 ⫾ 4.3
1.05 ⫾ 0.5
0.27 ⫾ 0.0
9.15 ⫾ 0.3
15.25 ⫾ 0.2
9.26 ⫾ 1.1
9.65 ⫾ 0.1
2.36 ⫾ 0.2
1.2 ⫾ 0.0
1.34
1.85
0.63
0.81
2.25
4.44
* Values are the mean ⫾ SEM relative change in mean fluorescence
intensity of the positive stained probe minus the control antibody for
untreated (baseline) and tumor necrosis factor ␣ (TNF␣)–treated
cells. The relative change in expression (RCE) of 52-kd Ro/SSA
protein was calculated from protein expression in treated cells divided
by protein expression in untreated cells as described in Materials and
Methods (values ⬎1 ⫽ up-regulation; values ⬍1 ⫽ down-regulation).
Figure 3. Example of the cytometric analysis of the inhibitory effect
of neutralizing monoclonal antibodies against tumor necrosis factor ␣
receptor I (anti-TNFRI) on the up-regulation of intracellular 52-kd
Ro/SSA protein expression in primary human keratinocytes cultured
in the presence of tumor necrosis factor ␣ (TNF␣) for 24 hours. The
observed up-regulation was inhibited by pretreating the cells with
anti-TNFRI. Results for a representative sample are shown. MFI ⫽
mean fluorescence intensity; PE ⫽ phycoerythrin.
not shown). Like flow cytometry, Western blotting revealed variable degrees of constitutive 52-kd Ro/SSA
protein expression and up-regulation.
TNF␣-induced 52-kd Ro/SSA up-regulation in
primary human keratinocytes is mediated by TNFRI
signaling and inhibited by TNFRII signaling. To define
the roles of TNFRI and TNFRII in TNF␣-induced
up-regulation of 52-kd Ro/SSA autoantigen expression,
we first reproduced the constitutive surface expression
of both TNF␣ receptor subunits. These findings confirmed those of previous studies (32,33). Neutralizing
monoclonal antibodies directed against TNFRI inhibited TNF␣-induced up-regulation of intracellular 52-kd
Ro/SSA mRNA and protein expression, whereas antiTNFRII antibodies enhanced TNF ␣ -induced upregulation of 52-kd Ro/SSA protein expression (Figures
3 and 4). Treatment of the cells with a combination of
anti-sTNFRI and anti-sTNFRII in the presence of
TNF␣ resulted in the strongest inhibition of 52-kd
Ro/SSA mRNA expression.
Significant apoptosis in human keratinocytes not
induced by TNF␣. Flow cytometric analysis of primary
human keratinocytes stained with annexin V revealed
UP-REGULATION OF 52-kd Ro/SSA BY TNF␣ VIA TNFRI
Figure 4. Anti-TNFRI antibodies inhibit TNF ␣ -induced upregulation of intracellular 52-kd Ro/SSA mRNA and protein expression in primary human keratinocytes from different donors. The cells
were cultured with TNF␣ in the presence or absence of neutralizing
monoclonal antibodies directed against TNFRI (␣R⌱) and/or TNFRII
(␣R⌱⌱). Neutralization of TNFRII enhanced the up-regulatory effect
of TNF␣. Expression of 52-kd Ro/SSA autoantigen was calculated in
relation to GAPDH for mRNA and in terms of the MFI for protein.
The mRNA values represent the mean and SEM of triplicate measurements, and the protein values are the mean and SEM of duplicate
measurements. Messenger RNA and protein expression were determined using cells from different donors. The results were reproduced
in 3 serial experiments. See Figure 3 for definitions.
the presence of apoptotic cells in all untreated in vitro
cultures at 24 hours. The degree of apoptosis varied
widely (mean ⫾ SEM 39.8 ⫾ 6.9% [range 13–76%]; n ⫽
10). After incubating the cells with TNF␣ for up to 24
hours, no significant enhancement of apoptosis was
found in 6 of 10 samples, and an antiapoptotic effect was
detected in 1 of 10 samples. In 3 samples, expansion of
the apoptotic cell fraction was observed after cultivation
with TNF␣. The mean ⫾ SEM rate of apoptosis in
TNF␣-treated keratinocytes was 40.8 ⫾ 6.8% (range
16–87%; n ⫽ 10). The TNF␣-treated cells and untreated
controls did not differ significantly with regard to apoptosis.
DISCUSSION
Since previous investigations have shown that the
proinflammatory cytokine TNF␣ enhances surface expression of 52-kd Ro/SSA (24), the present study aimed
to clarify the effect of TNF␣ on the regulation of the
52-kd Ro/SSA autoantigen. The present study is the first
to demonstrate that TNF␣ is able to induce the upregulation of 52-kd Ro/SSA mRNA expression in hu-
535
man keratinocytes, and that this effect is followed by an
increase in 52-kd Ro/SSA synthesis at the protein level.
In contrast to 52-kd Ro/SSA surface expression, which
appeared within a maximum of 2 hours, mRNA was
up-regulated within 4 hours followed by an intracellular
protein increase 24 hours after TNF␣ treatment. This
suggests that different mechanisms might underlie antigen expression and translocation and that these processes can occur independently of each other. However,
the consequences of the up-regulation of intracellular
autoantigen expression and its role in pathogenesis have
yet to be investigated.
Consistent with reported findings, our data lead
us to speculate that TNF␣ might be a mediator of the
anti-Ro/SSA antibody–associated lesions occurring in
photosensitive cutaneous lupus. UV exposure triggers
the release of TNF␣ from keratinocytes (25,26) and
mast cells (27). The extent to which TNF␣ secretion can
be induced by other factors or other pathways is unknown. The enhancement of 52-kd Ro/SSA expression
in keratinocytes may contribute to the development of
cutaneous lesions through ADCC (21–23) or
complement-dependent cytotoxicity (34).
SCLE patients tend to develop such autoimmune
pathologies, as reflected by the higher susceptibility of
keratinocytes from lupus patients to UV irradiation–
induced ADCC compared with that of keratinocytes
from normal controls (21). Among other factors, this
may be related to the strong association of the ⫺308A
polymorphism of the TNF␣ promoter with SLE (35,36)
and SCLE (37). The ⫺308A polymorphism was recently
shown to be associated with increased TNF␣ accumulation in primary fibroblast cultures and skin from SCLE
patients. Other factors may also contribute to TNF␣
production, since SCLE cells secrete more TNF␣ than
healthy cells, even when matched for TNF␣ genotype
(38).
Further, there are indications that keratinocytes
derived from lupus patients with photosensitive cutaneous lesions may express more 52-kd Ro/SSA than keratinocytes from healthy donors (34,39). Therefore, it is
also conceivable that keratinocytes from lupus patients
are more susceptible to TNF␣ in terms of mRNA and
protein expression of 52-kd Ro/SSA. In our study,
human keratinocytes from healthy donors exhibited a
broad variability of baseline and TNF␣-induced Ro/SSA
expression. Further investigation of the TNF␣ effect on
52-kd Ro/SSA expression in keratinocytes from SLE
patients would be of interest to determine whether this
higher susceptibility might be a part of the pathogenetic
mechanism in cutaneous lupus lesions.
536
Consequently, TNF␣ blockade might be an effective approach to treating the skin lesions associated with
photosensitive cutaneous lupus. If TNF␣ blocking
agents are effective in treating photosensitive cutaneous
lupus erythematosus lesions, this could confirm that
TNF␣ plays a role in the pathogenesis of these lesions.
Infliximab, a monoclonal anti-TNF␣ antibody, did in
fact lead to an impressive response in one of our
patients, a woman with SCLE that was refractory to all
conventional drugs (40).
Up-regulation of 52-kd and 60-kd Ro/SSA
mRNA and protein expression in response to estradiol,
which also plays a role in lupus, has already been
demonstrated in human keratinocytes and in human
cancer cells of the MCF-7 line (41). Since UVB radiation triggers the release of TNF␣, it could lead to
up-regulation of Ro/SSA mRNA expression as well.
However, Szegedi et al could not find any changes in
Ro/SSA mRNA expression in keratinocytes of the HaCaT cell line that were exposed to UV irradiation (42).
The most probable explanation for this discrepancy with
our results is that Szegedi et al measured mRNA
expression after 24–72 hours of UVB radiation, which
was too late for detection of changes in Ro/SSA mRNA.
Our results clearly demonstrate that the up-regulation of
52-kd Ro/SSA is a fast and transient response, peaking
at 4 hours and no longer detectable after 8 hours. The
fact that we used the real-time PCR technique that was
sensitive enough to detect slight changes in weakly
expressed genes (⬍2.5 ⫻ 102 molecules) such as 52-kd
Ro/SSA is another important difference.
TNF␣ signaling is mediated by both TNFRI,
which bears an intracellular death domain, and TNFRII.
The present study revealed that the up-regulation of
52-kd Ro/SSA by TNF␣ is mediated by TNFRI signaling
and inhibited by TNFRII signaling.
Internalization of TNF␣ –TNFR complexes,
which has been shown to occur when the ligand binds to
keratinocytes, could theoretically be responsible for the
transient up-regulation of 52-kd Ro/SSA mRNA (43),
because this could lead to termination of TNF␣ signaling and degradation of Ro/SSA mRNA. However, we
demonstrated that short-term up-regulation of the autoantigen was sufficient for induction of the protein, which
was detectable within 24 hours of TNF␣ treatment.
Although the mean up-regulation of mRNA was relatively high, only slight up-regulation of the protein was
observed in different donors. One could speculate that
expression of the protein is regulated at the translational
level or that mRNA is degraded and not all molecules
are translated. However, because cell material was lim-
GERL ET AL
ited, we were unable to analyze 52-kd Ro/SSA expression in keratinocytes from the same donor at both the
transcriptional and translational levels.
TNF␣ activates several intracellular pathways
that can lead to either apoptosis or cell survival. There is
evidence suggesting that TNFRII can influence apoptotic signaling, which is mainly promoted by TNFRI. This
was demonstrated in T lymphocytes and Jurkat cells in
which TNF␣-induced caspase activation mediated by
stimulation of TNFRI was potentiated by stimulation
with TNFRII (44).
TNF␣-specific apoptosis did not occur in most of
our samples. Thus, the regulatory intracellular pathway
of TNF␣-mediated modulation of 52-kd Ro/SSA expression occurs via TNFRI without the induction of apoptosis. Although both processes are regulated by the same
molecules at the receptor level, they may have different
intracellular regulatory mechanisms. The unresponsiveness of human keratinocytes to TNF␣ with regard to
apoptosis induction has already been shown (33,45). In
another study, the investigators postulated that TNF␣mediated apoptosis is counteracted by constitutively
expressed antiapoptotic proteins (46).
TNFRI-induced apoptosis has been shown to
include the activation of FADD/MORT1 (47–49), which
in turn recruits FLICE/caspase 8 (47,50). This cysteine
protease triggers a proteolytic cascade and the activation
of downstream caspases. Another pathway induced by
TNF␣ includes the activation of human NF-␬B (51), a
factor that activates the transcription of various genes.
In knockout mice, NF-␬B was shown to be a downstream
effector that plays a critical role in protecting cells from
TNF␣-induced apoptosis (52). This pathway could be
responsible for the unresponsiveness of human keratinocytes to TNF␣-induced apoptosis and may also play a
role in the apoptosis-independent regulation of 52-kd
Ro/SSA expression seen in our experiments.
In conclusion, TNF␣ is able to up-regulate 52-kd
Ro/SSA expression in human keratinocytes via TNFRI
and to inhibit it via TNFRII. This suggests a possible
involvement of TNF␣ in the pathogenesis of anti-Ro/
SSA–associated cutaneous lupus lesions, which will be
elucidated in further experiments. TNF␣ targeting
should therefore be considered when developing future
strategies for the treatment of photosensitive cutaneous
lupus.
REFERENCES
1. Dorner T, Held C, Trebeljahr G, Lukowsky A, Yamamoto K,
Hiepe F. Serologic characteristics in primary biliary cirrhosis
UP-REGULATION OF 52-kd Ro/SSA BY TNF␣ VIA TNFRI
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
associated with sicca syndrome. Scand J Gastroenterol 1994;29:
655–60.
Dorner T, Feist E, Pruss A, Chaoui R, Goldner B, Hiepe F.
Significance of autoantibodies in neonatal lupus erythematosus.
Int Arch Allergy Immunol 2000;123:58–66.
Magro CM, Crowson AN. The cutaneous pathology associated
with seropositivity for antibodies to SSA (Ro): a clinicopathologic
study of 23 adult patients without subacute cutaneous lupus
erythematosus. Am J Dermatopathol 1999;21:129–37.
Von Muhlen CA, Tan EM. Autoantibodies in the diagnosis of
systemic rheumatic diseases. Semin Arthritis Rheum 1995;24:
323–58.
Sontheimer RD, Thomas JR, Gilliam JN. Subacute cutaneous
lupus erythematosus: a cutaneous marker for a distinct lupus
erythematosus subset. Arch Dermatol 1979;115:1409–15.
Sontheimer RD, Maddison PJ, Reichlin M, Jordon RE, Stastny P,
Gilliam JN. Serologic and HLA associations in subacute cutaneous
lupus erythematosus, a clinical subset of lupus erythematosus. Ann
Intern Med 1982;97:664–71.
Kephart DC, Hood AF, Provost TT. Neonatal lupus erythematosus: new serologic findings. J Invest Dermatol 1981;77:331–3.
Lee LA, Gaither KK, Coulter SN, Norris DA, Harley JB. Pattern
of cutaneous immunoglobulin G deposition in subacute cutaneous
lupus erythematosus is reproduced by infusing purified anti-Ro
(SSA) autoantibodies into human skin-grafted mice. J Clin Invest
1989;83:1556–62.
Van Venrooij WJ, Slobbe RL, Pruijn GJ. Structure and function of
La and Ro RNPs. Mol Biol Rep 1993;18:113–9.
Furukawa F, Kashihara-Sawami M, Lyons MB, Norris DA. Binding of antibodies to the extractable nuclear antigens SS-A/Ro and
SS-B/La is induced on the surface of human keratinocytes by
ultraviolet light (UVL): implications for the pathogenesis of
photosensitive cutaneous lupus. J Invest Dermatol 1990;94:77–85.
Golan TD, Elkon KB, Gharavi AE, Krueger JG. Enhanced
membrane binding of autoantibodies to cultured keratinocytes of
systemic lupus erythematosus patients after ultraviolet B/ultraviolet A irradiation. J Clin Invest 1992;90:1067–76.
Jones SK. Ultraviolet radiation (UVR) induces cell-surface Ro/
SSA antigen expression by human keratinocytes in vitro: a possible
mechanism for the UVR induction of cutaneous lupus lesions. Br J
Dermatol 1992;126:546–53.
LeFeber WP, Norris DA, Ryan SR, Huff JC, Lee LA, Kubo M, et
al. Ultraviolet light induces binding of antibodies to selected
nuclear antigens on cultured human keratinocytes. J Clin Invest
1984;74:1545–51.
Furukawa F, Lyons MB, Lee LA, Coulter SN, Norris DA. Estradiol enhances binding to cultured human keratinocytes of antibodies specific for SS-A/Ro and SS-B/La: another possible mechanism
for estradiol influence of lupus erythematosus. J Immunol 1988;
141:1480–8.
Furukawa F, Imamura S, Norris DA. Stimulation of anti-RNP
antibody binding to cultured keratinocytes by estradiol. Arch
Dermatol Res 1991;283:258–61.
Furukawa F, Ikai K, Matsuyoshi N, Shimizu K, Imamura S.
Relationship between heat shock protein induction and the binding of antibodies to the extractable nuclear antigens on cultured
human keratinocytes. J Invest Dermatol 1993;101:191–5.
Zhu J. Cytomegalovirus infection induces expression of 60 KD/Ro
antigen on human keratinocytes. Lupus 1995;4:396–406.
Saegusa J, Kawano S, Koshiba M, Hayashi N, Kosaka H, Funasaka
Y, et al. Oxidative stress mediates cell surface expression of
SS-A/Ro antigen on keratinocytes. Free Radic Biol Med 2002;32:
1006–16.
Zhang J, Xu Z, Jin J, Zhu T, Ma S. Induction of Ro/SSA antigen
expression on keratinocyte cell membrane by heat shock and
phorbol 12-myristate 13-acetate as well as estradiol and ultraviolet
B. J Dermatol Sci 2000;24:92–8.
537
20. Casciola-Rosen LA, Anhalt G, Rosen A. Autoantigens targeted in
systemic lupus erythematosus are clustered in two populations of
surface structures on apoptotic keratinocytes. J Exp Med 1994;
179:1317–30.
21. Furukawa F, Itoh T, Wakita H, Yagi H, Tokura Y, Norris DA, et
al. Keratinocytes from patients with lupus erythematosus show
enhanced cytotoxicity to ultraviolet radiation and to antibodymediated cytotoxicity. Clin Exp Immunol 1999;118:164–70.
22. Norris DA, Ryan SR, Fritz KA, Kubo M, Tan EM, Deng JS, et al.
The role of RNP, Sm, and SS-A/Ro-specific antisera from patients
with lupus erythematosus in inducing antibody-dependent cellular
cytotoxicity (ADCC) of targets coated with nonhistone nuclear
antigens. Clin Immunol Immunopathol 1984;31:311–20.
23. Yoshimasu T, Hiroi A, Ohtani T, Uede K, Furukawa F. Comparison of anti 60 and 52 kDa SS-A/Ro antibodies in the pathogenesis
of cutaneous lupus erythematosus. J Dermatol Sci 2002;29:35–41.
24. Dorner T, Hucko M, Mayet WJ, Trefzer U, Burmester GR, Hiepe
F. Enhanced membrane expression of the 52 kDa Ro(SS-A) and
La(SS-B) antigens by human keratinocytes induced by TNF␣. Ann
Rheum Dis 1995;54:904–9.
25. Avalos-Diaz E, Alvarado-Flores E, Herrera-Esparza R. UV-A
irradiation induces transcription of IL-6 and TNF␣ genes in
human keratinocytes and dermal fibroblasts. Rev Rhum Engl Ed
1999;66:13–9.
26. Kock A, Schwarz T, Kirnbauer R, Urbanski A, Perry P, Ansel JC,
et al. Human keratinocytes are a source for tumor necrosis factor
␣: evidence for synthesis and release upon stimulation with
endotoxin or ultraviolet light. J Exp Med 1990;172:1609–14.
27. Walsh LJ. Ultraviolet B irradiation of skin induces mast cell
degranulation and release of tumour necrosis factor-␣. Immunol
Cell Biol 1995;73:226–33.
28. Rheinwald JG, Green H. Serial cultivation of strains of human
epidermal keratinocytes: the formation of keratinizing colonies
from single cells. Cell 1975;6:331–43.
29. Itoh K, Itoh Y, Frank MB. Protein heterogeneity in the human
Ro/SSA ribonucleoproteins: the 52- and 60-kD Ro/SSA autoantigens are encoded by separate genes. J Clin Invest 1991;87:177–86.
30. Riemekasten G, Marell J, Trebeljahr G, Klein R, Hausdorf G,
Haupl T, et al. A novel epitope on the C-terminus of SmD1 is
recognized by the majority of sera from patients with systemic
lupus erythematosus. J Clin Invest 1998;102:754–63.
31. Karawajew L, Ruppert V, Wuchter C, Kosser A, Schrappe M,
Dorken B, et al. Inhibition of in vitro spontaneous apoptosis by
IL-7 correlates with bcl-2 up-regulation, cortical/mature immunophenotype, and better early cytoreduction of childhood T-cell
acute lymphoblastic leukemia. Blood 2000;96:297–306.
32. Neuner P, Pourmojib M, Klosner G, Trautinger F, Knobler R.
Increased release of the tumour necrosis factor receptor p75 by
immortalized human keratinocytes results from an activated shedding mechanism and is not related to augmented steady-state
levels of p75 mRNA. Arch Dermatol Res 1996;288:691–6.
33. Schwarz A, Bhardwaj R, Aragane Y, Mahnke K, Riemann H,
Metze D, et al. Ultraviolet-B-induced apoptosis of keratinocytes:
evidence for partial involvement of tumor necrosis factor-␣ in the
formation of sunburn cells. J Invest Dermatol 1995;104:922–7.
34. Yu HS, Chiang LC, Chang CH, Kang JW, Yu CL. The cytotoxic
effect of neonatal lupus erythematosus and maternal sera on
keratinocyte cultures is complement-dependent and can be augmented by ultraviolet irradiation. Br J Dermatol 1996;135:
297–301.
35. Rudwaleit M, Tikly M, Khamashta M, Gibson K, Klinke J, Hughes
G, et al. Interethnic differences in the association of tumor
necrosis factor promoter polymorphisms with systemic lupus erythematosus. J Rheumatol 1996;23:1725–8.
36. Wilson AG, Gordon C, di Giovine FS, de Vries N, van de Putte
LB, Emery P, et al. A genetic association between systemic lupus
538
37.
38.
39.
40.
41.
42.
43.
44.
GERL ET AL
erythematosus and tumor necrosis factor ␣. Eur J Immunol
1994;24:191–5.
Werth VP, Zhang W, Dortzbach K, Sullivan K. Association of a
promoter polymorphism of tumor necrosis factor-␣ with subacute
cutaneous lupus erythematosus and distinct photoregulation of
transcription. J Invest Dermatol 2000;115:726–30.
Werth VP, Kwon EJ, Bashir M, Sullivan KE, Patel P, Zhang W.
Endogenous overproduction of TNF␣ in photosensitive autoimmune diseases [abstract]. Arthritis Rheum 2003;48 Suppl
9:S598.
Ioannides D, Golden BD, Buyon JP, Bystryn JC. Expression of
SS-A/Ro and SS-B/La antigens in skin biopsy specimens of patients with photosensitive forms of lupus erythematosus. Arch
Dermatol 2000;136:340–6.
Hiepe F, Bruns A, Feist E, Burmester GR. Successful treatment of
a patient suffering from a refractory subacute cutaneous lupus
erythematosus (SCLE) with blockers of tumour necrosis factor A
[abstract]. Arthritis Rheum 2004;50 Suppl 9:S413.
Wang D, Chan EK. 17-␤-estradiol increases expression of 52-kDa
and 60-kDa SS-A/Ro autoantigens in human keratinocytes and
breast cancer cell line MCF-7. J Invest Dermatol 1996;107:610–4.
Szegedi A, Irinyi B, Bessenyei B, Marka M, Hunyadi J, Semsei I.
UVB light and 17-␤-estradiol have different effects on the mRNA
expression of Ro/SSA and La/SSB autoantigens in HaCaT cells.
Arch Dermatol Res 2001;293:275–82.
Pillai S, Bikle DD, Eessalu TE, Aggarwal BB, Elias PM. Binding and
biological effects of tumor necrosis factor ␣ on cultured human
neonatal foreskin keratinocytes. J Clin Invest 1989;83:816–21.
Chan FK, Lenardo MJ. A crucial role for p80 TNF-R2 in
amplifying p60 TNF-R1 apoptosis signals in T lymphocytes. Eur
J Immunol 2000;30:652–60.
45. Tsuru K, Horikawa T, Budiyanto A, Hikita I, Ueda M, Ichihashi
M. Low-dose ultraviolet B radiation synergizes with TNF-␣ to
induce apoptosis of keratinocytes. J Dermatol Sci 2001;26:209–16.
46. Reinartz J, Bechtel MJ, Kramer MD. Tumor necrosis factor-␣induced apoptosis in a human keratinocyte cell line (HaCaT) is
counteracted by transforming growth factor-␣. Exp Cell Res
1996;228:334–40.
47. Boldin MP, Mett IL, Varfolomeev EE, Chumakov I, Shemer-Avni
Y, Camonis JH, et al. Self-association of the “death domains” of
the p55 tumor necrosis factor (TNF) receptor and Fas/APO1
prompts signaling for TNF and Fas/APO1 effects. J Biol Chem
1995;270:387–91.
48. Chinnaiyan AM, O’Rourke K, Tewari M, Dixit VM. FADD, a
novel death domain-containing protein, interacts with the death
domain of Fas and initiates apoptosis. Cell 1995;81:505–12.
49. Kischkel FC, Hellbardt S, Behrmann I, Germer M, Pawlita M,
Krammer PH, et al. Cytotoxicity-dependent APO-1 (Fas/CD95)associated proteins form a death-inducing signaling complex
(DISC) with the receptor. EMBO J 1995;14:5579–88.
50. Muzio M, Chinnaiyan AM, Kischkel FC, O’Rourke K, Shevchenko
A, Ni J, et al. FLICE, a novel FADD-homologous ICE/CED-3-like
protease, is recruited to the CD95 (Fas/APO-1) death-inducing
signaling complex. Cell 1996;85:817–27.
51. Meyer R, Hatada EN, Hohmann HP, Haiker M, Bartsch C,
Rothlisberger U, et al. Cloning of the DNA-binding subunit of
human nuclear factor ␬ B: the level of its mRNA is strongly
regulated by phorbol ester or tumor necrosis factor ␣. Proc Natl
Acad Sci U S A 1991;88:966–70.
52. Beg AA, Baltimore D. An essential role for NF-␬B in preventing
TNF-␣-induced cell death. Science 1996;274:782–4.
Документ
Категория
Без категории
Просмотров
3
Размер файла
136 Кб
Теги
necrosis, factors, autoantigen, ross, intracellular, keratinocyte, regulated, receptov, via, tumors
1/--страниц
Пожаловаться на содержимое документа