вход по аккаунту


Down-regulation of FLIP sensitizes rheumatoid synovial fibroblasts to Fas-mediated apoptosis.

код для вставкиСкачать
Vol. 50, No. 9, September 2004, pp 2803–2810
DOI 10.1002/art.20453
© 2004, American College of Rheumatology
Down-Regulation of FLIP Sensitizes Rheumatoid Synovial
Fibroblasts to Fas-Mediated Apoptosis
Guillermo Palao, Begoña Santiago, Marı́a Galindo, Mónica Payá,
Juan C. Ramirez, and José L. Pablos
not detected. TNF␣ induced increases in FLIPL and
FLIPS expression and protected RA FLS from apoptosis, while CHX induced the opposite effects. Downregulation of FLIP by antisense oligonucleotide strongly
sensitized RA FLS to Fas-mediated apoptosis.
Conclusion. Apoptosis susceptibility and FLIP
expression are similar in OA and RA FLS. Downregulation of FLIP sensitizes RA FLS to Fas-mediated
apoptosis and may be a valuable tool for targeting RA
FLS hyperplasia.
Objective. Hyperplasia of fibroblast-like synoviocytes (FLS) contributes to chronic inflammation and
joint destruction in rheumatoid arthritis (RA). FLICEinhibitory protein (FLIP) is an antiapoptotic protein
that might prevent apoptotic elimination of FLS in
response to death ligands such as tumor necrosis factor
␣ (TNF␣) or Fas ligand, which are present in RA
synovium. Previous studies on FLIP expression by osteoarthritis (OA) and RA FLS have shown variable
results, and the specific role of FLIP as an apoptosis
inhibitor in these cells remains unclear. We undertook
this study to investigate the expression and antiapoptotic function of FLIP in FLS.
Methods. We studied the expression of FLIP by
immunohistochemistry and immunoblotting in synovial
tissues or cultured FLS from RA and OA patients. FLS
apoptosis was induced by an agonistic anti-Fas monoclonal antibody and FLS were then quantified. We
studied the effects of cycloheximide (CHX), TNF␣, and
FLIP antisense oligonucleotide on FLIP expression and
FLS apoptotic susceptibility.
Results. FLIPL was the isoform mainly expressed
in lining synoviocytes and cultured FLS. Synovial tissues and cultured FLS from OA and RA tissues displayed similar patterns and levels of expression of FLIP.
Fas-induced apoptosis was variable in different FLS
lines, but differences between OA and RA groups were
Inflammatory cell infiltration and the expansion
of an aggressive population of fibroblast-like synoviocytes (FLS) in the synovial membrane are the pathologic
hallmarks of rheumatoid arthritis (RA). FLS overgrowth
may result from unbalanced proliferation and apoptosis,
and both processes have been detected on tissue sections
of rheumatoid synovium (1–3). Since proliferation is
scarce in RA synovium (4), dysregulation of apoptosis
has been proposed to explain the accumulation of FLS
(5). In RA synovium, despite the expression of several
death receptor ligands (such as tumor necrosis factor ␣
[TNF␣] and Fas ligand [FasL]) by infiltrating cells in
close proximity to FLS, the net effect is toward hyperplasia rather than apoptotic elimination of FLS (5–7).
Rheumatoid FLS display increased expression of several
antiapoptotic proteins, but their specific function in
these cells has not been completely elucidated (8–10).
Whether cultured RA FLS are different from osteoarthritis (OA) FLS regarding susceptibility to Fasmediated apoptosis remains controversial (11–13).
The response of different cell types to ligands of
death receptors may vary from cell proliferation or
activation to cell death, depending upon the different
signaling pathways induced in each particular cell type
(14). The first step induced by activation of death
receptors is the recruitment of a complex of different
proteins termed DISC (death-inducing signaling com-
Supported by grants 02/0057 and G03/152 from Fondo de
Investigación Sanitaria (Spain). Dr. Palao’s work was supported by
Fondo de Investigación Sanitaria. Dr. Santiago’s work was supported
by a grant from Fundación Española de Reumatologı́a–Abbott Laboratories.
Guillermo Palao, MD, Begoña Santiago, PhD, Marı́a Galindo, MD, Mónica Payá, MSc, Juan C. Ramirez, PhD, José L. Pablos,
MD: Hospital 12 de Octubre, Madrid, Spain.
Address correspondence and reprint requests to José L.
Pablos, MD, Servicio de Reumatologı́a, Hospital 12 de Octubre,
Avenida Andalucı́a s.n., 28041 Madrid, Spain. E-mail: jlpablos@h12o.
es or
Submitted for publication January 26, 2004; accepted in
revised form May 5, 2004.
plex) to the intracellular death domain of these receptors through the adapter FADD (15). DISC formation
results in the homodimerization and proteolytic activation of caspase 8, and this step is counterbalanced by the
parallel recruitment of FLICE-inhibitory protein
(FLIP), which hetero-oligomerizes with caspase 8 and
reduces its proteolytic activation (16,17). Caspase 8 may
induce apoptosis in RA FLS through direct activation of
terminal caspases or by prior activation of caspase 9
through the mitochondrial apoptotic pathway. Specific
inhibition of Fas signaling by inhibitors of caspase 8,
caspase 9, or terminal caspases efficiently restrains Fasmediated RA FLS apoptosis (12,18). However, although
FLIP has been demonstrated to be a dominant inhibitor
of caspase 8 activation and Fas-induced apoptosis in a
large variety of human cells (19,20), its differential
expression and potential in preventing Fas-mediated
apoptosis of rheumatoid FLS are still matters of debate.
On the one hand, studies of the expression of
FLIP in cultured OA and RA FLS have shown variable
expression in different cell lines, but not disease-specific
significant differences (10,21). This may relate to FLIP
heterogeneity among cells from different synovial regions, as suggested by in situ hybridization studies which
have shown preferential expression of FLIP in areas of
bone and cartilage invasion (10). On the other hand,
enforced FLIP expression has been shown to partially
decrease Fas-induced apoptosis in RA FLS (22), but the
function of constitutively expressed FLIP as an apoptosis
inhibitor has not been evaluated in RA FLS.
We have studied the expression of FLIP protein
in synovial tissues and in cultured FLS from RA and OA
patients, and we have examined its relevance in the
susceptibility of these cells to Fas-induced apoptosis. We
show that disease-specific differences in the expression
of FLIP or in the susceptibility of FLS to Fas-mediated
apoptosis do not occur; however, the constitutive expression of FLIP protein by FLS is an important survival
factor that decreases the susceptibility of this cell type to
Fas-induced apoptosis.
Induction of apoptosis in FLS cultures. Synovial FLS
were cultured by explant growth from synovial tissue obtained
from RA or OA patients undergoing knee replacement surgery. All patients fulfilled the 1987 American College of
Rheumatology (formerly, the American Rheumatism Association) revised criteria for the classification of RA (23). Cells
were cultured in 10% fetal calf serum–Dulbecco’s modified
Eagle’s medium in plastic flasks. FLS from 8 OA and 9 RA
patients were used between passages 3 and 6.
Fas stimulation was performed by exposing cells to the
Fas-activating anti-Fas IgM monoclonal antibody (mAb)
CH11 at 1 ␮g/ml for 24 hours (MBL, Nagoya, Japan). This
decavalent IgM antibody induces Fas receptor crosslinking
similar to that of membrane FasL, in contrast to soluble FasL
(24), which in our preliminary studies did not induce FLS
apoptosis even in the presence of cycloheximide (CHX).
Where indicated, 1.5 ␮g/ml CHX was simultaneously added at
the time of anti-Fas treatment. To inhibit caspase activation,
cells were preincubated with the cell-permeable inhibitor
Z-VAD-FMK at 20 ␮M (Biomol, Plymouth Meeting, PA) 30
minutes before adding the anti-Fas CH11 mAb. Where indicated, cells were pretreated with human TNF␣ at 50 ng/ml (BD
PharMingen, San Diego, CA) for 8 hours before adding
anti-Fas mAb.
Cell death and apoptosis assays. Cell death induced by
exposure to anti-Fas mAb for 24 hours was quantified by direct
counting of live and dead cells in Neubauer chambers, after
staining with 0.2% trypan blue. All dead cells were quantified
after recovery of both trypsinized adherent cells and previously
detached cells, floating in the media, by centrifugation. To
quantify apoptotic cell death, mono- and oligonucleosomal
DNA was determined in FLS cytoplasmic extracts by enzymelinked immunosorbent assay (ELISA) according to the manufacturer’s protocol (Roche Diagnostics, Mannheim, Germany).
In preliminary experiments, apoptosis was confirmed by
TUNEL fluorescent labeling of FLS grown on coverslips as
previously reported (19), which showed a good correlation with
results of nucleosomal release ELISAs.
Western blot analysis. Protein from 106 FLS was
extracted in ice-cold lysis buffer (10 mM Tris HCl [pH 8.0], 1
mM EDTA, 150 mM NaCl, 0.1% sodium dodecyl sulfate, 10
␮g/ml leupeptin, 10 ␮g/ml aprotinin, 2 ␮g/ml pepstatin A, and
0.5 mM phenylmethylsulfonyl fluoride). Protein extracts (30
␮g) were electrophoresed on 10% polyacrylamide gel and
electrophoretically transferred to nitrocellulose filters. After
blocking for 2 hours with 5% nonfat dried milk in Tris buffered
saline containing 0.05% Tween 20 (TBST), the membranes
were incubated overnight at 4°C with 1:500 anti-FLIP (Alexis,
Lausen, Switzerland), 1:250 anti-FADD (Transduction Laboratories, Lexington, KY), 1:700 anti–caspase 8 (MBL), or
1:6,000 anti–␤-actin (clone AC-15; Sigma-Aldrich Quı́mica,
Madrid, Spain) antibodies in 5% nonfat dried milk–TBST. The
filters were washed and incubated for 1 hour with secondary
antibodies linked to peroxidase at 1:8,000 dilution. Bands were
visualized by an enhanced chemiluminescence system (Pierce,
Rockford, IL) and analyzed by densitometry.
Immunohistochemistry. Frozen sections from the
above-described RA or OA synovial tissue samples were fixed
in cold acetone for 10 minutes and, after blocking in 5% serum,
were incubated overnight at 4°C with primary antibody. We
used two different anti-FLIP antibodies, a polyclonal antibody
(BD PharMingen) which preferentially recognized the FLIPS
isoform in our Western blot studies, and mAb G-11 (Santa
Cruz Biotechnology, Santa Cruz, CA), which preferentially
recognized the FLIPL isoform. Negative controls with mouse
IgG or nonimmune rabbit serum were included.
Immunoperoxidase detection was performed by the
ABC method according to the instructions of the manufacturer
(Vector, Burlingame, CA). Peroxidase activity was developed
Figure 1. Expression of FLICE-inhibitory protein (FLIP) by immunohistochemistry in synovial tissue sections
from patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Frozen sections were immunostained with
antibodies preferentially recognizing FLIPL (a and b) or FLIPS (d and e) isoforms. RA (a and d) and OA (b and
e) tissue sections are representative of 9 and 8 synovial tissue samples, respectively. Controls were performed by
incubating sections with mouse (c) or rabbit (f) IgG instead of anti-FLIP antibodies. Immunoperoxidase-labeled
cells appear brown, and sections are counterstained with hematoxylin. (Original magnification ⫻ 400 in a–f; ⫻
1,000 in insets.)
by diaminobenzidine substrate, and slides were counterstained
in Gills hematoxylin.
Antisense (AS) oligodeoxynucleotide transfection. A
phosphorothioate-modified and fluorescein isothiocyanate
(FITC)–conjugated single-stranded oligodeoxynucleotide directed against the human FLIP translation initiation codon
(FLIP-AS: 5⬘-GACTTCAGCAGACATCCTAC-3⬘) and a similarly modified control nonsense (NS) oligodeoxynucleotide
(FLIP-NS: 5⬘-TGGATCC GACATGTCAGA-3⬘) were synthesized as previously described by Perlman et al (20). RA FLS
were grown to ⬃50% confluence and incubated with 5 ␮M
FITC-conjugated oligodeoxynucleotides premixed with Oligofectamine (Life Technologies, Gaithersburg, MD). Transfection efficiency was analyzed 24 hours later by flow cytometry.
After 4 hours of incubation with the transfection mixture,
anti-Fas antibody CH11 was added and cells were analyzed for
apoptosis at 24 hours. FLIP protein expression in transfected
cells was analyzed by Western blot in parallel cultures 24 hours
after transfection.
Expression of FLIP protein in OA and RA FLS.
FLIP protein was uniformly detected in the synovial
lining and in rare scattered cells of the sublining of RA
and OA synovial tissue sections by anti-FLIPL antibody,
and differences between groups were not observed
(Figures 1a–c). Since previous studies have focused on
macrophage staining of FLIP, and this cell type has been
shown to mainly express FLIPS (25) (in contrast to
fibroblasts, which mainly express FLIPL [19]), we also
immunostained with an anti-FLIP antibody which in our
laboratory preferentially recognizes FLIPS by Western
blot. Immunostaining with this antibody showed a different pattern, consisting of abundant immunostained
cells with large mononuclear, macrophage-like morphology in the sublining of both RA and OA tissues
(Figures 1d–f). In RA tissue sections, a higher number of
FLIP-positive macrophage-like cells was detected (results not shown). In contrast, lining synoviocytes were
not immunostained by this antibody.
To further analyze FLIP isoforms expressed by
FLS and potential differences between RA and OA
FLS, we performed Western blot studies in cultured
cells. Cultured FLS from either RA or OA synovium
displayed abundant expression of FLIPL isoform in
Western blots (Figure 2a), whereas FLIPS was only
barely detected (results not shown). Expression of
FLIPL was variable in different OA and RA cell lines,
but a densitometric analysis corrected by ␤-actin expression did not detect significant differences between OA
and RA groups (Figure 2b).
Since caspase 8 levels may also modify apoptotic
susceptibility in response to Fas activation in other
human fibroblasts (19), we also examined caspase 8
protein expression by OA and RA FLS lines. Similar to
did not modify caspase 8 or FADD protein expression
(Figure 3a).
We also analyzed the effect of TNF␣ treatment
on FLIP protein expression in RA FLS. Treatment with
TNF␣ increased the expression levels of FLIPL and
FLIPS isoforms, with a maximal increase at 8 hours
(Figure 3b).
Fas-mediated apoptosis in OA and RA FLS. Fas
triggering by exposure to CH11 mAb for 24 hours
induced detectable apoptosis in all RA and OA FLS
lines, as evaluated by cytotoxicity or nucleosomal release
assays (Figure 4). Treatment with CHX significantly
sensitized cultured RA and OA FLS to Fas-induced
apoptosis (P ⬍ 0.005). Although Fas- and Fas ⫹ CHX–
mediated apoptosis were variable in different FLS lines,
statistically significant differences between OA and RA
groups were not detected (Figure 4).
Pretreatment of FLS with the caspase inhibitor
Z-VAD-FMK prevented apoptosis in response to either
anti-Fas alone or anti-Fas ⫹ CHX (Figure 4c). Pretreatment of RA FLS with TNF␣ for 8 hours did not induce
detectable apoptosis, but it significantly decreased the
apoptotic response to anti-Fas alone or to anti-Fas ⫹
CHX (Figure 4d).
Apoptosis and down-regulation of FLIP by antisense oligonucleotide. Since CHX significantly increased
the susceptibility to apoptosis of cultured FLS and
Figure 2. Expression of FLIP and caspase 8 in RA and OA fibroblastlike synoviocytes (FLS). Protein extracts from FLS lines obtained from
different patients were analyzed by Western blot. The immunoblot
shown is representative of 3 independent experiments including all
FLS lines from 9 different RA and 8 different OA donors (a). Results
of densitometric analysis are shown (b), including mean and SD levels
of FLIP, caspase 8, or FLIP:caspase 8 ratio normalized to ␤-actin
protein expression in 8 OA and 9 RA FLS lines. See Figure 1 for other
the findings for FLIP, different cell lines displayed
variable levels of caspase 8 expression, but significant
differences between RA and OA FLS lines were not
detected (Figure 2). The FLIP:caspase 8 ratio was also
similar in both groups.
To analyze whether the main intracellular proteins involved in Fas signaling were specifically modulated by low concentrations of CHX in RA FLS, we
treated RA FLS with CHX at a 10-fold lower concentration than that required to inhibit protein synthesis
nonspecifically. Treatment of RA FLS with CHX abrogated FLIP protein expression, whereas this treatment
Figure 3. Effect of cycloheximide (CHX) and tumor necrosis factor ␣
(TNF␣) on the expression of Fas pathway proteins by RA fibroblastlike synoviocytes (FLS). RA FLS were incubated for 24 hours with
either 1.5 ␮g/ml CHX or medium alone, and FLIP, FADD, caspase 8,
and ␤-actin protein expression levels were analyzed by Western blot
(a). RA FLS were incubated with TNF␣ for different time periods, and
FLIP and ␤-actin protein expression levels were analyzed by Western
blot (b). Results are representative of 3 independent experiments
using different RA FLS lines. Unst. ⫽ unstimulated cells (see Figure
1 for other definitions).
Figure 4. Apoptosis of FLS in response to Fas activation. Cultured FLS were activated by anti-Fas CH11 monoclonal antibody for 24 hours in the
presence or absence of CHX as indicated. Apoptosis was quantified by cytotoxicity (a) or by nucleosomal release (b) assays in 8 OA and 9 RA FLS
lines. The effect of pretreatment with the caspase inhibitor Z-VAD-FMK (zVAD) on Fas-mediated apoptosis of RA FLS is also shown (c). Data
are representative of 3 independent experiments including 5 different RA FLS lines. Also shown is the effect of TNF␣ pretreatment of FLS on their
susceptibility to Fas-mediated apoptosis in the presence or absence of CHX (d). Data are representative of 3 independent experiments performed
in 5 different RA FLS lines. Values are the mean and SD. Abs ⫽ absorbance (see Figures 1 and 3 for other definitions).
down-regulated FLIP expression, whereas the opposite
effects were observed by treatment with TNF␣, we
evaluated whether specific down-regulation of FLIP
protein by FLIP-AS oligonucleotide modulates FLS
susceptibility to Fas. Transfection of RA FLS with
FLIP-AS or NS oligonucleotide was monitored by flow
cytometric studies of transfected cultures performed in
parallel to those used for analysis of FLIP protein
expression by Western blot and for Fas-induced apoptosis. This analysis demonstrated similar rates of FITCconjugated FLIP-AS or NS oligonucleotide transfection
(Figure 5a). By densitometric analysis of Western blots,
FLIP protein expression was down-regulated by 60% in
RA FLS transfected with FLIP-AS oligonucleotide compared with those transfected with NS oligonucleotide
(Figure 5b). Fas-induced apoptosis was increased 3-fold
in FLIP-AS–transfected RA FLS compared with NStransfected RA FLS (P ⬍ 0.001) (Figure 5c).
Our results confirm that FLIP protein is constitutively and similarly expressed by OA and RA FLS, in
which it operates as an inhibitor of Fas-mediated apoptosis, which explains the relative resistance of this cell
type to Fas activation. The kinetics of Fas-mediated cell
death in human fibroblasts are slow and inefficient
compared with those of other Fas-expressing cells, such
as lymphoid cells (19,26). This resistance to death
receptor–mediated apoptosis has also been observed in
other normal and tumor cells, and in many cases it is
abrogated by down-regulating FLIP protein, which is
very sensitive to low concentrations of CHX (27,28).
Fibroblasts from FLIP-knockout mice are more sensitive
to Fas-mediated apoptosis (29), and human dermal
fibroblasts are also sensitized to Fas activation by CHX
or FLIP antisense oligonucleotide transfection (19),
Figure 5. Effect of FLIP antisense (AS-FLIP) oligonucleotide transfection on FLIP expression and Fas-induced apoptosis. RA FLS were
transfected with fluorescein isothiocyanate (FITC)–conjugated ASFLIP or nonsense (NS) oligonucleotide, and uptake was analyzed by
flow cytometry (a). The open histogram depicts the fluorescence of
untransfected cells, the shaded histogram represents AS-FLIP–
transfected cells, and the superimposed thick-line histogram represents the fluorescence of NS-transfected cells. FLIP protein expression
was analyzed by Western blot after 24 hours of transfection (b). After
transfection, RA FLS were treated with anti-Fas CH11 monoclonal
antibody for 24 hours, and apoptosis was quantified by nucleosomal
release assay (c). Data are representative of 3 independent experiments using 3 different RA FLS lines. Values are the mean and SD.
Abs ⫽ absorbance (see Figures 1 and 3 for other definitions).
which points to constitutive FLIP as a relevant antiapoptotic factor in this cell type. In TNF␣-treated RA FLS,
adenoviral transfer of FLIP gene decreases their susceptibility to Fas-mediated apoptosis (22). However, the
effect of enforced FLIP expression does not reflect a
physiologic situation, and such expression may either
protect against or induce apoptosis depending upon the
level of expression achieved (30).
Our data demonstrate that most RA FLS survive
after 24 hours of Fas challenge, reflecting a relative
resistance compared with other Fas receptor–expressing
cell types. Down-regulation of constitutively expressed
FLIP by CHX or specific FLIP antisense oligonucleotide
strongly sensitizes these cells to Fas-mediated apoptosis.
This suggests that constitutively expressed FLIP oper-
ates as a dominant inhibitor of Fas-mediated apoptosis
in rheumatoid FLS.
The pattern of expression of FLIP proteins in
synovial tissues reveals differential expression of FLIPL
and FLIPS isoforms by synoviocytes and sublining macrophages. Whereas FLIPL is the main isoform observed in
cultured FLS and lining synoviocytes, we found FLIPS in
sublining large mononuclear cells, consistent with previous observations that identified mainly the FLIPS isoform in rheumatoid macrophages (25). We did not find
differences in the levels of expression of FLIPL between
cultured OA and RA FLS or in lining synoviocytes in
tissue sections.
A previous study of synovial tissue sections by
Catrina et al identified higher levels of FLIP in early RA
than in late RA; however, these observations seemed to
apply to sublining macrophages, in which these investigators preferentially detected FLIP protein expression
(31). Investigators in another study failed to detect FLIP
protein in protein extracts from either OA or RA FLS,
in contrast to findings in FLS obtained from joint
fractures; in that study, however, the isoform detected
was not specified (21). Schedel et al detected variable
FLIP expression in cultured OA and RA FLS lines
obtained at synovectomy or arthroplastic surgery, without significant differences between groups (10). Interestingly, using in situ hybridization, these investigators
identified higher FLIP messenger RNA expression in
fibroblasts located in areas of bone and cartilage erosion. Collectively, these data suggest that FLIP expression is similar in RA FLS and OA FLS, although
different FLS lines or fibroblasts located in different
areas can be heterogeneous.
Previous studies in other cell types have shown
that FLIP behaves as an NF-␬B–inducible factor (32,33).
Therefore, activation of NF-␬B by local exposure to
TNF␣ may increase FLIP expression and consequently
protect RA synoviocytes from Fas-mediated apoptosis.
Previous studies in cultured RA FLS have shown either
down- or up-regulation of FLIP after variable periods of
exposure to TNF␣ (10,22). Our data show that, consistent with the antiapoptotic role of FLIP, exposure to
TNF␣ increases FLIP expression and decreases Fasmediated apoptosis, suggesting another pathogenetic
mechanism for this pleiotropic cytokine in RA.
TNF␣ and Fas share several intracellular signaling pathways (14,15). On the one hand, both can induce
the assembling of the intracellular DISC, leading to
caspase 8 activation and apoptosis. FLIP protein can
also protect cells from TNF␣-mediated apoptosis (33),
and, consistently, RA FLS are completely resistant to
TNF␣-mediated apoptosis unless NF-␬B activation is
blocked (34). On the other hand, TNF␣ and Fas receptor activation induces NF-␬B translocation, which leads
to increased FLIP expression (35,36). This NF-␬B loop
of Fas signaling may protect cells from Fas-mediated cell
death, resulting in proinflammatory and survival, rather
than cytotoxic, effects of death receptor stimulation
(36–38). Permissivity to death receptor–induced apoptosis in other cell types has been suggested to depend upon
low NF-␬B and FLIP induction by death ligand activation (39). Our preliminary data show that Fas also
activates NF-␬B in RA FLS, further linking NF-␬B
activation and FLIP-mediated protection against apoptosis to rheumatoid FLS hyperplasia (40). These observations collectively suggest that FLIP protein is an
important regulator of death receptor signaling in RA
FLS, and this has obvious pathogenetic and therapeutic
implications in RA, in which death ligands are abundantly expressed by infiltrating cells.
1. Qu Z, Garcia CH, O’Rourke LM, Planck SR, Kohli M, Rosenbaum JT. Local proliferation of fibroblast-like synoviocytes contributes to synovial hyperplasia: results of proliferating cell nuclear
antigen/cyclin, c-myc, and nucleolar organizer region staining.
Arthritis Rheum 1994;37:212–20.
2. Firestein GS, Yeo M, Zvaifler NJ. Apoptosis in rheumatoid
arthritis synovium. J Clin Invest 1995;96:1631–8.
3. Matsumoto S, Muller-Ladner U, Gay RE, Nishioka K, Gay S.
Ultrastructural demonstration of apoptosis, Fas and Bcl-2 expression of rheumatoid synovial fibroblasts. J Rheumatol 1996;23:
4. Lalor PA, Mapp PI, Hall PA, Revell PA. Proliferative activity of
cells in the synovium as demonstrated by a monoclonal antibody,
Ki67. Rheumatol Int 1987;7:183–6.
5. Baier A, Meineckel I, Gay S, Pap T. Apoptosis in rheumatoid
arthritis. Curr Opin Rheumatol 2003;15:274–9.
6. Asahara H, Hasumuna T, Kobata T, Yagita H, Okumura K, Inoue H,
et al. Expression of Fas antigen and Fas ligand in the rheumatoid
synovial tissue. Clin Immunol Immunopathol 1996;81:27–34.
7. Deleuran BW, Chu CQ, Field M, Brennan FM, Mitchell T,
Feldmann M, et al. Localization of tumor necrosis factor receptors
in the synovial tissue and cartilage–pannus junction in patients
with rheumatoid arthritis: implications for local actions of tumor
necrosis factor ␣. Arthritis Rheum 1992;35:1170–8.
8. Perlman H, Georganas C, Pagliari LJ, Koch AE, Haines K III,
Pope RM. Bcl-2 expression in synovial fibroblasts is essential for
maintaining mitochondrial homeostasis and cell viability. J Immunol 2000;164:5227–35.
9. Franz JK, Pap T, Hummel KM, Nawrath M, Aicher WK,
Shigeyama Y, et al. Expression of sentrin, a novel antiapoptotic
molecule, at sites of synovial invasion in rheumatoid arthritis.
Arthritis Rheum 2000;43:599–607.
10. Schedel J, Gay RE, Kuenzler P, Seemayer C, Simmen B, Michel
BA, et al. FLICE-inhibitory protein expression in synovial fibroblasts and at sites of cartilage and bone erosion in rheumatoid
arthritis. Arthritis Rheum 2002;46:1512–8.
11. Nakajima T, Aono H, Hasunuma T, Yamamoto K, Shirai T,
Hirohata K, et al. Apoptosis and functional Fas antigen in
rheumatoid arthritis synoviocytes. Arthritis Rheum 1995;38:485–91.
Itoh K, Hase H, Kojima H, Saotome K, Nishioka K, Kobata T.
Central role of mitochondria and p53 in Fas-mediated apoptosis of
rheumatoid synovial fibroblasts. Rheumatology (Oxford) 2004;43:
Ichikawa K, Liu W, Fleck M, Zhang H, Zhao L, Ohtsuka T, et al.
TRAIL-R2 (DR5) mediates apoptosis of synovial fibroblasts in
rheumatoid arthritis. J Immunol 2003;171:1061–9.
Budd RC. Death receptors couple to both cell proliferation and
apoptosis. J Clin Invest 2002;109:437–41.
Ashkenazi A, Dixit VM. Death receptors: signaling and modulation. Science 1998;281:1305–8.
Krueger A, Baumann S, Krammer PH, Kirchhoff S. FLICEinhibitory proteins: regulators of death receptor-mediated apoptosis. Mol Cell Biol 2001;21:8247–54.
Irmler M, Thome M, Hahne M, Schneider P, Hofmann K, Steiner
V, et al. Inhibition of death receptor signals by cellular FLIP.
Nature 1997;388:190–5.
Okamoto K, Kobayashi T, Kobata T, Hasunuma T, Kato T,
Sumida T, et al. Fas-associated death domain protein is a Fasmediated apoptosis modulator in synoviocytes. Rheumatology
(Oxford) 2000;39:471–80.
Santiago B, Galindo M, Palao G, Pablos JL. Intracellular regulation of Fas-induced apoptosis in human fibroblasts by extracellular
factors and cycloheximide. J Immunol 2004;172:560–6.
Perlman H, Pagliari LJ, Georganas C, Mano T, Walsh K, Pope
RM. FLICE-inhibitory protein expression during macrophage
differentiation confers resistance to fas-mediated apoptosis. J Exp
Med 1999;190:1679–88.
Tolboom TC, Medema JP, van Gaalen FA, Pieterman E, Huizinga
TW, Toes RE. Fibroblast-like synoviocytes from rheumatoid arthritis patients express less FLICE-inhibitory protein than fibroblast-like synoviocytes from trauma patients: comment on the
article by Schedel et al [letter]. Arthritis Rheum 2003;48:858–9.
Kobayashi T, Okamoto K, Kobata T, Hasunuma T, Kato T,
Hamada H, et al. Differential regulation of Fas-mediated apoptosis of rheumatoid synoviocytes by tumor necrosis factor ␣ and basic
fibroblast growth factor is associated with the expression of
apoptosis-related molecules. Arthritis Rheum 2000;43:1106–14.
Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF,
Cooper NS, et al. The American Rheumatism Association 1987
revised criteria for the classification of rheumatoid arthritis.
Arthritis Rheum 1988;31:315–24.
Suda T, Hashimoto H, Tanaka M, Ochi T, Nagata S. Membrane
Fas ligand kills human peripheral blood T lymphocytes, and
soluble Fas ligand blocks the killing. J Exp Med 1997;186:2045–50.
Perlman H, Pagliari LJ, Liu H, Koch AE, Haines GK III, Pope
RM. Rheumatoid arthritis synovial macrophages express the
Fas-associated death domain–like interleukin-1␤–converting
enzyme–inhibitory protein and are refractory to Fas-mediated
apoptosis. Arthritis Rheum 2001;44:21–30.
Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, et
al. Two CD95 (APO-1/Fas) signaling pathways. EMBO J 1998;17:
Willems F, Amraoui Z, Vanderheyde N, Verhasselt V, Aksoy E,
Scaffidi C, et al. Expression of c-FLIP(L) and resistance to
CD95-mediated apoptosis of monocyte-derived dendritic cells:
inhibition by bisindolylmaleimide. Blood 2000;95:3478–82.
Fulda S, Meyer E, Debatin KM. Metabolic inhibitors sensitize for
CD95 (APO-1/Fas)-induced apoptosis by down-regulating Fasassociated death domain-like interleukin 1-converting enzyme
inhibitory protein expression. Cancer Res 2000;60:3947–56.
Yeh WC, Itie A, Elia AJ, Ng M, Shu HB, Wakeham A, et al.
Requirement for Casper (c-FLIP) in regulation of death receptor-
induced apoptosis and embryonic development. Immunity 2000;
Chang DW, Xing Z, Pan Y, Algeciras-Schimnich A, Barnhart BC,
Yaish-Ohad S, et al. c-FLIP(L) is a dual function regulator for
caspase-8 activation and CD95-mediated apoptosis. EMBO J
Catrina AI, Ulfgren AK, Lindblad S, Grondal L, Klareskog L. Low
levels of apoptosis and high FLIP expression in early rheumatoid
arthritis synovium. Ann Rheum Dis 2002;61:934–6.
Kreuz S, Siegmund D, Scheurich P, Wajant H. NF-␬B inducers
upregulate cFLIP, a cycloheximide-sensitive inhibitor of death
receptor signaling. Mol Cell Biol 2001;21:3964–73.
Micheau O, Lens S, Gaide O, Alevizopoulos K, Tschopp J. NF-␬B
signals induce the expression of c-FLIP. Mol Cell Biol 2001;21:
Zhang HG, Huang N, Liu D, Bilbao L, Zhang X, Yang P, et al.
Gene therapy that inhibits nuclear translocation of nuclear factor
␬B results in tumor necrosis factor ␣–induced apoptosis of human
synovial fibroblasts. Arthritis Rheum 2000;43:1094–105.
Kataoka T, Budd RC, Holler N, Thome M, Martinon F, Irmler M,
et al. The caspase-8 inhibitor FLIP promotes activation of NF-␬B
and Erk signaling pathways. Curr Biol 2000;10:640–8.
Ahn JH, Park SM, Cho HS, Lee MS, Yoon JB, Vilcek J, et al.
Non-apoptotic signaling pathways activated by soluble Fas ligand
in serum-starved human fibroblasts: mitogen-activated protein
kinases and NF-␬B-dependent gene expression. J Biol Chem
Wajant H, Pfizenmaier K, Scheurich P. Non-apoptotic Fas signaling. Cytokine Growth Factor Rev 2003;14:53–66.
Miagkov AV, Kovalenko DV, Brown CE, Didsbury JR, Cogswell
JP, Stimpson SA, et al. NF-␬B activation provides the potential
link between inflammation and hyperplasia in the arthritic joint.
Proc Natl Acad Sci U S A 1998;95:13859–64.
Micheau O, Tschopp J. Induction of TNF receptor I-mediated
apoptosis via two sequential signaling complexes. Cell 2003;114:
Pablos JL, Santiago B, Galindo M. Rheumatoid synoviocytes
display NF-kappaB activation and chemokines expression in response to Fas receptor activation [abstract]. Arthritis Rheum
2002;46 Suppl 9:S552.
Без категории
Размер файла
238 Кб
apoptosis, sensitized, regulation, synovial, flip, fas, rheumatoid, mediated, downs, fibroblasts
Пожаловаться на содержимое документа