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Inhibition of CD95 apoptotic signaling by interferon-╨Ю╤Ц in human osteoarthritic chondrocytes is associated with increased expression of FLICE inhibitory protein.

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ARTHRITIS & RHEUMATISM
Vol. 50, No. 2, February 2004, pp 498–506
DOI 10.1002/art.20008
© 2004, American College of Rheumatology
Inhibition of CD95 Apoptotic Signaling by Interferon-␥ in
Human Osteoarthritic Chondrocytes Is Associated With
Increased Expression of FLICE Inhibitory Protein
Francesco Grassi,1 Anna Piacentini,1 Sandra Cristino,1 Stefania Toneguzzi,1
Andrea Facchini,2 and Gina Lisignoli1
Objective. Cartilage homeostasis dysregulation
during osteoarthritis (OA) has been linked to an increased rate of apoptosis of chondrocytes, the only cell
type resident in the cartilage. In addition, the CD95–
CD95 ligand (the Fas system) has emerged as one of the
major pathways of cell death in the cartilage. We
undertook the present study to investigate the role of
interferon-␥ (IFN␥) in the regulation of the Fas system
by analyzing the modulation of intracellular signaling
molecules (FLICE inhibitory protein [FLIP] and
caspases 3 and 8) in primary cultures of human OA
chondrocytes.
Methods. CD95-induced apoptotic death of human OA chondrocytes was analyzed in the presence or
absence of IFN␥ using cell death immunoassay for
apoptosis, real-time polymerase chain reaction for FLIP
and caspase 8 expression, Western blotting for FLIP,
and proteolytic activity for caspases 3 and 8.
Results. CD95-induced apoptotic death of human
OA chondrocytes was strongly counteracted by IFN␥
treatment, although the surface expression of CD95 was
slightly up-regulated by this cytokine. The messenger
RNA (mRNA) expression of FLIP and caspase 8, mediators involved in CD95 signaling, revealed that FLIP
expression in human OA chondrocytes was significantly
up-regulated (2-fold increase) by IFN␥ treatment.
Moreover, the FLIP:caspase 8 mRNA ratio increased
significantly. FLIP up-regulation by IFN␥ was confirmed at the protein level. Caspase 8 and caspase 3
proteolytic activities, both induced in these cells by
stimulation with anti-CD95, were also significantly
down-modulated by IFN␥.
Conclusion. These findings suggest that IFN␥
impairs CD95-mediated signaling and apoptotic death
in human chondrocytes. Its mechanism of action involves down-regulation of caspase 8 and caspase 3
activities and increased expression of the antiapoptotic
protein FLIP, suggesting an interesting mechanism for
the inhibition of chondrocyte apoptosis.
Cartilage homeostasis dysregulation is a main
feature of the osteoarthritic (OA) joint. Following repetitive mechanical stress as well as altered genetic
factors, cartilage undergoes profound structural lesions
mainly due to an increased synthesis of catabolic cytokines and matrix-degrading proteases (1,2). Chondrocytes, the only cell type present in the cartilage, play an
essential regulatory function in matrix turnover. However, the rate of cellular turnover is affected during OA
progression. Evidence is accumulating that chondrocyte
response to mechanical and biochemical insults includes
an increased rate of apoptosis (3–5), thus resulting in
decreased tissue cellularity of the OA cartilage (6,7), an
increased number of empty lacunae, and abnormal
calcification of the subchondral bone (5,8). The hypothesis of a pathogenetic role of chondrocyte apoptosis in
OA is supported by studies showing in situ a higher
level of apoptosis in the OA cartilage compared with
normal tissue (4,5) and a linkage between the rate of
chondrocyte apoptosis and the severity of cartilage
Supported by MURST (60% fund), Ricerca Corrente IOR,
and the FP Health Ministry of Italy.
1
Francesco Grassi, PhD, Anna Piacentini, PhD, Sandra Cristino, PhD, Stefania Toneguzzi, BSc, Gina Lisignoli, PhD: Istituti
Ortopedici Rizzoli, Bologna, Italy; 2Andrea Facchini, MD: Istituti
Ortopedici Rizzoli, Bologna, Italy, and Università degli Studi di
Bologna, Bologna, Italy.
Address correspondence and reprint requests to Gina
Lisignoli, PhD, Laboratorio di Immunologia e Genetica, I.O.R., Via di
Barbiano 1/10, 40136 Bologna, Italy. E-mail: labimge@alma.unibo.it.
Submitted for publication February 19, 2003; accepted in
revised form October 13, 2003.
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INHIBITION OF APOPTOSIS BY IFN␥ IN HUMAN OA CHONDROCYTES
damage (4,9). Moreover, a number of mediators that
are overexpressed in the synovial fluid of OA patients,
such as nitric oxide, interleukin-17, and CD95 ligand
(CD95L), have the potential to induce chondrocyte
apoptosis (10,11).
CD95 (Fas/APO-1) and its ligand, CD95L, constitute the so-called Fas system, which represents a
major mechanism in the regulation of the homeostasis
of the immune cells and other cell types (i.e., tumor
cells) (12). CD95 engagement triggers a variety of
signaling molecules, including caspases, phospholipases,
and protein kinases. In particular, sequential caspase
activation results in the specific proteolytic events typical
of programmed cell death. Some caspases act as initiators (caspases 8 and 10), while some others act
as executioners (caspases 3, 6, and 7) (13). FLICE
inhibitory protein (FLIP), the enzymatically inactive
homolog of caspase 8, has been recently regarded as an
intriguing regulator of cellular sensitivity to apoptotic
cell death, due to its ability to block caspase 8 recruitment and activation at the death-inducing signaling
complex (DISC) level (14,15). In particular, the FLIPL
(long) isoform seems to be a more potent inhibitor than
the FLIPS (short) isoform at comparable expression
levels (14). In different cell types, including synovial
macrophages and fibroblasts obtained from patients
with arthritis, the level of FLIP expression has been
correlated with cellular resistance to apoptosis induced
by CD95 (16–21).
A pivotal regulatory effect of interferon-␥ (IFN␥)
on CD95 expression and function has been demonstrated in other cell systems, leading in most cases to an
up-regulation of the CD95-dependent apoptotic death
(22–24). Among cytokines and growth factors, which are
implicated in the regulation of cartilage homeostasis, the
role of IFN␥ is still poorly clarified. In vitro studies have
demonstrated relevant effects of IFN␥ on chondrocyte
metabolism (25,26) and a protective effect against cartilage breakdown (27). However, no data are available
regarding the possible regulatory effect of IFN␥ on the
CD95-mediated pathway of cell death in cartilage.
In the present in vitro study, we investigated the
role of IFN␥ in CD95-mediated cell death in human OA
chondrocytes. We demonstrated that CD95-induced apoptotic death of human OA chondrocytes is strongly
counteracted by IFN␥ treatment. The antiapoptotic
effect of IFN␥ is associated with decreased enzymatic
activity of both caspase 8 and caspase 3. Finally, we
demonstrated an increased expression of FLIP leading
to an increased FLIP:caspase 8 messenger RNA
499
(mRNA) ratio in treated chondrocytes, thus suggesting
an intriguing role for IFN␥ in cartilage pathophysiology.
MATERIALS AND METHODS
Reagents. Recombinant human IFN␥ and tumor necrosis factor ␣ (TNF␣) were purchased from Roche Molecular
Biochemicals (Mannheim, Germany). Penicillin/streptomycin,
L-glutamine, and cycloheximide (CHX) were purchased from
Sigma (St. Louis, MO). Anti-CD95 antibodies were purchased
from Medical and Biological Laboratories (Nagoya, Japan)
(clone CH-11) and Ancell (Bayport, MN), respectively, for
functional tests and flow cytometric analysis of surface receptor expression. Anti–type I collagen and anti–type II collagen
antibodies were obtained from Chemicon International (Temecula, CA). Dulbecco’s modified Eagle’s medium (DMEM)
was obtained from Life Technologies (Gaithersburg, MD), and
fetal calf serum (FCS) was purchased from Mascia Brunelli
(Milan, Italy).
Specimens and cell culture. Human OA cartilage was
obtained from 8 patients who were undergoing total joint
replacement of the knee. Diagnosis of OA was based on
clinical, laboratory, and radiographic evaluation. The study
was approved by the Institutional Review Board. Chondrocytes
were obtained by sequential enzymatic digestion of minced
fragments of cartilage, as previously described (28). Briefly, the
isolated chondrocytes were filtered with nylon meshes, seeded
in culture flasks, and grown in DMEM supplemented with 10%
FCS, L-glutamine, and antibiotics. Cells were used after the
first or second passage of culture. Immunocytochemical assessment of the chondrocyte phenotype was performed using
monoclonal antibodies (mAb) against type I or type II collagen. For all experiments described in this report, chondrocytes
were seeded at a density of 6 ⫻ 104/cm2.
Fluorescence-activated cell sorting analysis. Chondrocytes from 6 donors with OA were assessed for CD95 expression by flow cytometry. Chondrocytes were seeded in 24-well
plates for 48 hours and then treated or not treated with IFN␥
and TNF␣ (100 units/ml) for 24 hours. Cells were then
trypsinized, preincubated with human Ig (2 mg/ml in phosphate buffered saline [PBS] with 2% FCS and 0.1% sodium
azide) for 30 minutes at 4°C, washed twice in PBS, and
incubated either with anti-CD95 mAb diluted 1:500 or with
IgG1 isotype control for 30 minutes at 4°C. Chondrocytes were
washed with PBS and incubated with fluorescein
isothiocyanate–conjugated rabbit anti-mouse IgG (diluted
1:25; Dako, Glostrup, Denmark) for 30 minutes at 4°C. Finally,
cells were fixed in 2% paraformaldehyde and analyzed by
FACStar Plus (Becton Dickinson, Sunnyvale, CA).
Cell death detection immunoassay. For quantification
of nucleosomal fragment enrichment, chondrocytes were
seeded in 24-well plates. After 48 hours, IFN␥ and TNF␣ were
added to the culture medium at a final concentration of 100
units/ml for 24 hours. In dose-response experiments, IFN␥ was
added to the culture medium at final concentrations of 10
units/ml, 100 units/ml, and 500 units/ml for 24 hours. The
medium was then replaced, anti-CD95 mAb (clone CH-11) or
isotype-matched mouse IgM control mAb was added at a
concentration of 0.75 ␮g/ml, and the cells were incubated for a
further 14 hours at 37°C. At the end of the incubation time, the
500
cells were lysed, and fragmented nucleosomal DNA was measured using the photometric enzyme immunoassay (Cell Death
Detection ELISA Plus; Roche Molecular Biochemicals) according to the manufacturer’s instructions. Apoptosis induction was calculated as follows: [A405 nm (treated) ⫺ A405 nm
(untreated)]/A405 nm (untreated), where A ⫽ absorbance.
Real-time polymerase chain reaction (PCR) for FLIP
and caspase 8 expression. To analyze FLIP and caspase 8
expression at the mRNA level, chondrocytes were seeded in
12-well plates for 48 hours, then treated with IFN␥, anti-CD95,
or the combination of both, as indicated above. Total RNA
was isolated by the RNAzol B method (Biotecx Laboratories,
Houston, TX) and reverse transcribed using Moloney murine
leukemia virus reverse transcriptase and oligo(dT) priming
according to the manufacturer’s protocol (Perkin-Elmer Cetus,
Norwalk, CT). The following primers were designed for
complementary DNA (cDNA) amplification: FLIP, forward
5⬘-TGGACCTTGTGGTTGAGTTG-3⬘ and reverse 5⬘TTGGATTGCTGCTTGGAGA-3⬘ (179-bp product, including
both FLIPL and FLIPS isoforms; GenBank accession no.
U97074); caspase 8, forward 5⬘-AGAGCCTGAGAGAGCGATG-3⬘ and reverse 5⬘-CACCATCAATCAGAAGGGAAG-3⬘ (166-bp product; GenBank accession no. NM 001228).
Primers for the housekeeping gene GAPDH, used as an
internal control, were as previously described (29). All primers
were chosen to lie in different exons or to span exon junctions
to prevent amplification of genomic DNA.
Real-time PCR was run in a LightCycler Instrument
(Roche Molecular Biochemicals) using the QuantiTect SYBR
Green PCR kit (Qiagen, Hilden, Germany) with the following
protocol: initial activation of HotStar Taq DNA polymerase at
95°C for 15 minutes, 40 cycles of 94°C for 15 seconds, 58°C for
15 seconds, and 72°C for 10 seconds. To determine absolute
mRNA copy numbers, standard curves were generated for
FLIP, caspase 8, and GAPDH using 10-fold dilution series of
gel-purified PCR products (30). Two microliters of either
external standards (ranging from 6 ⫻ 106 to 6 ⫻ 101 copies) or
diluted cDNA samples (corresponding to 4 ng of total RNA
per sample) was amplified in separate tubes for each target
gene. The increase in PCR product was monitored for each
amplification cycle by measuring the increase in fluorescence
caused by the binding of SYBR Green I dye to doublestranded DNA. The crossing point values (i.e., the cycle
number at which the detected fluorescence exceeds the threshold value) were determined for each sample, and specificity of
the amplicons was confirmed by melting curve analysis.
Amplification efficiencies, as calculated from the
slopes of log input amounts plotted versus crossing point
values, were confirmed to be high (⬎90%) and comparable for
both target genes (⬍4% difference with respect to GAPDH).
Subsequently, to check for intersample variations (e.g., during
RNA extraction and/or reverse transcription), FLIP and
caspase 8 mRNA levels were normalized to the housekeeping
gene, using the calculated copy numbers.
Western blotting. Total cellular protein extracts were
obtained by washing the cells twice in PBS and resuspending
them in lysis buffer (0.5% Triton X-100, 300 mM NaCl, 50 mM
Tris HCl, pH 7.6, containing 1 mM phenylmethylsulfonyl
fluoride, 2 ␮g/ml aprotinin, and 10 ␮g/ml leupeptin). Cells
GRASSI ET AL
were kept on ice for 30 minutes and then centrifuged at 10,000g
for 10 minutes. The amount of cellular protein present in the
clarified supernatant was evaluated using the bicinchoninic
acid protein assay (Pierce, Rockford, IL). Equal amounts of
cellular protein (20 ␮g) from each sample were then separated
by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to Immobilon-P membranes (Millipore, Bedford, MA) using standard procedures. Blots were
hybridized with rat anti-human FLIP (clone Dave 3, recognizing both FLIPL and FLIPS isoforms; Alexis Biochemicals,
Lausen, Switzerland) or mouse anti–human ␤-actin antibody
(Sigma), followed by horseradish peroxidase–conjugated antirat IgG (Alexis Biochemicals) or anti-mouse IgG (Amersham
Biosciences, Little Chalfont, UK) antibody. Proteins were
visualized using SuperSignal West Pico substrate (Pierce).
Caspase 8 activity assay. Proteolytic activity of caspase
8 was evaluated using a FLICE/Caspase-8 Fluorometric Protease Assay Kit (Chemicon International) according to the
manufacturer’s instructions. For each experimental point,
1.2 ⫻ 106 chondrocytes were seeded for 48 hours and stimulated with IFN␥, anti-CD95, or the combination of both, as
described above. At the end of the incubation time, cells were
lysed and incubated with the fluorogenic substrate 7-amino-4trifluoromethyl coumarin for 2 hours at 37°C in a buffer
containing 5 mM dithiothreitol. Cells were kept under serumfree culture conditions in order to minimize the background
level of enzymatic activity. In blocking experiments, the protein synthesis inhibitor CHX was added to cell culture at a final
concentration of 10 ␮g/ml 1 hour before IFN␥ treatment and
maintained all during the incubation time. Samples were then
analyzed using a Spectra Max Fluorometer (Molecular Devices, Sunnyvale, CA) equipped with a 400-nm excitation filter
and a 505-nm emission filter.
Caspase 3 activity assay. For the determination of
caspase 3–specific activity, a fluorometric immunosorbent enzyme assay (Caspase 3 Activity Assay; Roche Molecular
Biochemicals) was used according to the manufacturer’s instructions. For each experimental point, 1.2 ⫻ 106 chondrocytes were plated and stimulated with IFN␥, anti-CD95, or the
combination of both, as described above. At the end of the
incubation time, cells were washed twice with cold PBS, lysed
for 1 minute on ice, and spun down for 15 minutes at 1,500g.
One hundred microliters of the lysate was then transferred into
a microtiter plate coated with anti–caspase 3 antibody and
incubated for 1 hour at 37°C. The plate was washed and
incubated for 2 hours at 37°C with 100 ␮l of substrate solution
containing the caspase 3–specific substrate Acetyl-Asp-GluVal-Asp-7-amido-4-trifluoromethyl-coumarin. Finally, the resulting fluorescence was measured at 505 nm in a Spectra Max
Fluorometer (Molecular Devices).
Analysis of CD95L in the synovial fluid of OA and
rheumatoid arthritis (RA) patients. The concentrations of
CD95L in the synovial fluid of 20 OA patients (mean ⫾ SD age
65 ⫾ 10 years, mean ⫾ SD disease duration 6 ⫾ 4 years) and
20 RA patients (mean ⫾ SD age 63 ⫾ 15 years, mean ⫾ SD
disease duration 12 ⫾ 8 years) were analyzed by sandwich
enzyme-linked immunosorbent assay (MBL, Tokyo, Japan)
according to the manufacturer’s instructions.
INHIBITION OF APOPTOSIS BY IFN␥ IN HUMAN OA CHONDROCYTES
501
RESULTS
Figure 1. Flow cytometry analysis of CD95 expression on human
chondrocytes obtained from patients with osteoarthritis (OA). CD95
expression was evaluated under basal conditions (untreated) and after
stimulation for 24 hours with 100 units/ml of tumor necrosis factor ␣
(TNF␣) or 100 units/ml of interferon-␥ (IFN␥). a, Flow cytometry
histograms from 1 of 5 representative experiments. Open histograms
represent isotype control. Shaded histograms represent CD95-specific
antibody. x-axis ⫽ channel numbers; y-axis ⫽ number of events. b,
Columns represent the mean ⫾ SD fluorescence intensity of 6 OA
chondrocyte samples analyzed by flow cytometry under basal conditions and after TNF␣ or IFN␥ treatment as described above.
Statistical analysis. The Wilcoxon test was used to
compare experimental groups. The analyses were performed
using CSS Statistica statistical software (StatSoft, Tulsa, OK).
Effect of IFN␥ and TNF␣ on CD95 surface
expression. To determine whether IFN␥ and TNF␣
regulate the expression of CD95 in human OA chondrocytes, 6 of 8 OA chondrocyte samples were analyzed
by flow cytometry for the surface expression of CD95.
As shown in Figures 1a and b, untreated human OA
chondrocytes expressed CD95 on their surface membranes. Incubation of cells for 24 hours with IFN␥ or
TNF␣ at a concentration of 100 units/ml resulted in a
slight up-regulation of CD95 expression.
Down-regulation of CD95-mediated apoptosis by
IFN␥. To assess the functional relevance of the CD95
modulation, we treated chondrocytes from 8 OA patients with anti-CD95 mAb (clone CH-11). Microscopic
phase-contrast studies showed that the addition of antiCD95 induced significant morphologic changes compared with untreated conditions. In particular, several
cells acquired smaller size morphology and rounded
shape and showed membrane blebbing. Moreover, a
small portion of the cells detached from the flask and
floated in the culture medium. When IFN␥ was added
24 hours before anti-CD95 treatment, the number of
cells undergoing such modification was much smaller.
To evaluate the amount of cellular apoptosis
under these conditions, we quantified nucleosomal fragment enrichment. As shown in Figure 2a, anti-CD95
treatment largely induced chondrocyte apoptosis. In
contrast, when cells were treated with IFN␥, CD95induced apoptosis was significantly inhibited (P ⫽
0.007). Interestingly, when chondrocytes were treated
with TNF␣ (100 units/ml), the rate of apoptosis induction did not change significantly compared with that in
the anti-CD95–treated cells. Dose-response experiments
revealed that the antiapoptotic effect of IFN␥ was dose
dependent (Figure 2b), and near-maximum inhibition
was achieved at a concentration of 100 units/ml, while
considerable inhibition was also observed at a concentration of 10 units/ml.
IFN␥-induced increase in FLIP production by
human chondrocytes. FLIP overexpression has been
recently correlated with the suppression of Fas signaling
in different cell types. Thus, chondrocytes from 6 OA
patients were analyzed for total FLIP expression at the
mRNA level, using real-time PCR. As shown in Figure
3a, our findings confirmed that human chondrocytes
express FLIP mRNA. Absolute quantification revealed
that in unstimulated chondrocytes, there was a mean ⫾
SD of 6,400 ⫾ 1,300 FLIP mRNA copies per 4 ng of
total RNA. Interestingly, cell stimulation with IFN␥ at
502
GRASSI ET AL
Figure 2. Effect of IFN␥ and TNF␣ on CD95-mediated DNA fragmentation. Analysis was performed on human OA chondrocytes after
treatment with IFN␥ alone or after treatment with anti-CD95, IFN␥ ⫹
anti-CD95, or TNF␣ ⫹ anti-CD95. Fragmented DNA was measured
by cell death detection immunoassay. a, Columns represent the
mean ⫾ SD of 8 OA chondrocyte samples analyzed. Data are
expressed as fold apoptosis induction calculated as follows: [A405 nm
(treated) ⫺ A405 nm (untreated)]/A405 nm (untreated), where A ⫽
absorbance. ⴱ ⫽ P ⫽ 0.007 versus anti-CD95 alone. b, Results of 1
representative dose-response experiment (of 3 performed), showing
the effect of different concentrations of IFN␥ (10 units/ml, 100
units/ml, and 500 units/ml) on CD95-mediated DNA fragmentation.
Values are the mean and SD. See Figure 1 for definitions.
100 units/ml induced a statistically significant (P ⫽ 0.02)
increase of FLIP mRNA expression, both in the presence and in the absence of anti-CD95 mAb. The rate of
increase was 2-fold higher than that in unstimulated
cells. Caspase 8 mRNA was also slightly increased with
IFN␥ treatment (Figure 3b); finally, the FLIP:caspase 8
mRNA ratio underwent a statistically significant increase (Figure 3c) in samples treated with IFN␥, both in
the presence (P ⫽ 0.02) and in the absence (P ⫽ 0.04) of
anti-CD95 mAb.
To determine whether the increased FLIP
mRNA expression resulted in higher protein levels,
immunoblotting for FLIPL was performed in samples
from 3 OA patients. Although FLIPS (75 kd) was
undetectable, the level of FLIPL (50 kd) protein ex-
Figure 3. Effect of interferon-␥ (IFN␥) on FLICE inhibitory protein
(FLIP) and caspase 8 mRNA expression. Real-time polymerase chain
reaction experiments for a, FLIP and b, caspase 8 were performed on
6 osteoarthritic (OA) chondrocyte samples under basal conditions
(untreated) and after treatment with IFN␥, anti-CD95, or IFN␥ ⫹
anti-CD95. Data are expressed as calculated mean ⫾ SD copy
numbers after normalization to the housekeeping gene GAPDH. For
FLIP mRNA copies, differences between values for untreated and
IFN␥-treated samples and between values for anti-CD95–treated and
IFN␥ ⫹ anti-CD95–treated samples were significant (ⴱ ⫽ P ⫽ 0.02). c,
Columns represent the mean ⫾ SD FLIP:caspase 8 ratios for the 6 OA
chondrocyte samples analyzed under the conditions indicated above. ⴱ
⫽ P ⫽ 0.04 versus untreated samples; ⴱⴱ ⫽ P ⫽ 0.02 versus samples
treated with anti-CD95 alone.
INHIBITION OF APOPTOSIS BY IFN␥ IN HUMAN OA CHONDROCYTES
pressed by OA chondrocytes was consistent with PCR
data, being higher in IFN␥-treated samples and very low
or undetectable in unstimulated cells (Figure 4). Thus,
inhibition of CD95-dependent apoptosis by IFN␥ is
associated with increased expression of FLIP, suggesting
a role for this caspase 8 antagonist in blocking the CD95
transduction pathway.
Inhibition of caspase 8 and caspase 3 proteolytic
activity inhibited by IFN␥. The antiapoptotic effect of
IFN␥ led us to further investigate the mechanism by
which this cytokine may interfere with CD95-mediated
apoptosis. Therefore, 6 of 8 OA samples were analyzed
for the proteolytic activity of caspase 8 and caspase 3,
since a recent study has demonstrated that these proteases are specifically activated after CD95 engagement
in human chondrocytes (31). As shown in Figures 5a and
b, respectively, both caspase 8 and caspase 3 were
significantly activated in samples treated for 14 hours
with anti-CD95 mAb (P ⫽ 0.043 for both caspases),
while treatment with IFN␥ resulted in a significant
down-modulation of these apoptotic proteases (P ⫽
0.043). Of interest, the effect of IFN␥ treatment on
caspase activation resembled that shown in Figure 2a for
nucleosomal fragment enrichment.
Effect of CHX on IFN␥-mediated inhibition of
caspase 8 activity. To determine whether IFN␥-induced
inhibition of caspase 8 activity requires new protein
synthesis, proteolytic activity of this enzyme was evaluated in isolated OA chondrocytes pretreated with the
protein synthesis inhibitor CHX at 10 ␮g/ml. As shown
Figure 4. Effect of IFN␥ on FLIP protein. Western blot analysis for
FLIP was performed on 3 OA chondrocyte samples under basal
conditions (lane 1) and after treatment with IFN␥ (lane 2), anti-CD95
(lane 3), or IFN␥ ⫹ anti-CD95 (lane 4). One of 3 representative
immunoblots is shown. FLIP-L ⫽ long isoform of FLIP (see Figure 3
for other definitions).
503
Figure 5. Effect of IFN␥ on caspase 8 (a) and caspase 3 (b) proteolytic activity. The analysis was performed on 6 OA chondrocyte
samples under basal conditions (untreated) and after treatment with
IFN␥, anti-CD95, or IFN␥ ⫹ anti-CD95. Data are expressed as
mean ⫾ SD fluorescence units. ⴱ ⫽ P ⫽ 0.043 versus samples treated
with anti-CD95 alone; ⴱⴱ ⫽ P ⫽ 0.043 versus untreated samples. See
Figure 3 for definitions.
in Figure 6, CHX treatment completely reversed the
inhibitory effect exerted by IFN␥, thus restoring the
level of caspase 8 activation observed in the presence of
anti-CD95 mAb alone.
Levels of CD95L in the synovial fluid of OA
and RA patients. To establish the physiologic relevance of our in vitro data, we analyzed the concentration of CD95L in the synovial fluid of OA and RA
patients. As shown in Figure 7, synovial fluid from OA
patients contained significant amounts of CD95L. The
levels of CD95L in the synovial fluid of RA patients
were significantly higher than the corresponding
levels in OA patients, as reported by other investigators
(32,33).
DISCUSSION
The progression of OA disease seems to be
closely related to the balance between life and death of
504
Figure 6. Necessary role of protein synthesis for interferon-␥ (IFN␥)–
mediated down-regulation of caspase 8 enzymatic activity. Results
shown are from 1 representative experiment (of 3 performed), showing
the effect of cycloheximide (CHX; 10 ␮g/ml) on caspase 8 proteolytic
activity. Data are expressed as mean and SD fluorescence units.
chondrocytes, the only cell type resident inside the
cartilage. One of the potential mechanisms by which
chondrocyte homeostasis is regulated is through the
CD95–CD95L pathway. We investigated the effect of
IFN␥ on anti-CD95–induced apoptosis in human OA
chondrocytes. We found that chondrocytes treated with
IFN␥ at 100 units/ml are largely resistant to apoptosis
induced by anti-CD95 mAb (CH-11).
In accord with previous results from other investigators, we found that human OA chondrocytes at first
passage in culture significantly express CD95 on their
surfaces. In contrast, chondrocytes do not seem to
produce CD95L by themselves (11). However, significant amounts of this molecule were found by us and
other investigators (32,33) in the synovial fluid of OA
and RA patients, thus leading us to consider the CD95
pathway as a mechanism relevant to cellular death in the
cartilage.
Despite the fact that surface expression of CD95
was slightly up-regulated in IFN␥-treated cells, CD95dependent apoptosis was largely decreased, thus emphasizing the relevance of the antiapoptotic effect of IFN␥.
This novel finding seems to be cell-type specific, since
several reports based on other cell systems provide
general evidence for a facilitating effect of this cytokine
on CD95-triggered apoptosis (23,24,34). To our knowledge, up to now only one report has documented a
delaying effect of IFN␥ (when added to cells at 1,000
units/ml) on CD95-dependent apoptosis in mature erythroid colony-forming cells (35). The specificity of our
data was also confirmed by the evaluation of 3 different
molecules involved in CD95 signaling.
FLIP, also known as FLICE inhibitory protein, is
GRASSI ET AL
a cytoplasmic protein which prevents the processing and
release of active caspase 8 from the receptor, thus acting
as a dominant-negative inhibitor of caspase 8 (15).
Human chondrocytes are already known to constitutively express FLIP mRNA (36). Here we report that
chondrocytes treated with IFN␥ display an increased
expression of cellular FLIP, at both the mRNA and
protein levels. Increased transcription of FLIP mRNA
has been shown to correlate well with apoptosis inhibition (20), demonstrating that FLIP is mainly regulated at
the transcriptional level. Therefore, to further investigate the relevance of IFN␥-dependent induction of
FLIP expression, we have used real-time PCR to calculate the number of mRNA copies for FLIP and caspase
8 in OA chondrocytes, thus focusing on whether IFN␥
affects the ratio of these two molecules.
Unstimulated cells expressed a mean ⫾ SD of
6,400 ⫾ 1,300 FLIP mRNA copies per 4 ng of total
RNA; this amount should be considered a fairly high
level if compared with the results of similar analyses
performed in other cell systems (18), and might explain
the slow kinetic of CD95-dependent apoptotic death
observed in human chondrocytes by us and others. The
level of caspase 8 mRNA expression was surprisingly low
in our cells (Figure 3b), nearly 10-fold lower than that of
FLIP expression (Figure 3a). The amount of caspase 8
mRNA did not change after anti-CD95 stimulation,
although the proteolytic activity under the same conditions increased significantly (Figure 5a). This finding
may be explained by the evidence that caspase 8 is
mostly regulated at the posttranscriptional level, and
perhaps the duration of the anti-CD95 stimulation in
our model might not be sufficient to induce an increased
transcription of the caspase 8 DNA.
Figure 7. Concentrations of CD95 ligand (CD95L) in synovial fluid of
patients with osteoarthritis (OA) and rheumatoid arthritis (RA). Each
data point represents 1 patient.
INHIBITION OF APOPTOSIS BY IFN␥ IN HUMAN OA CHONDROCYTES
Treatment of chondrocytes with IFN␥ led to a
statistically significant increase in the FLIP:caspase 8
mRNA ratio. Although several reports have correlated
the increased expression of FLIP to a reduced sensitivity
to CD95-mediated apoptosis, the amount of FLIP
needed to block this death pathway is not known.
Therefore, the increased FLIP:caspase 8 ratio that we
have found is likely to be biologically relevant for
explaining the reduced sensitivity of chondrocytes to
CD95-dependent apoptosis.
Although caspase 8 was undetectable at the protein level in our chondrocyte lysates, these findings are
supported by our data on FLIP analyzed at the protein
level. Immunoblotting of cultured chondrocytes revealed very low or undetectable levels of FLIPL in
unstimulated cells; however, protein expression was
clearly increased by IFN␥ treatment, even in the presence of anti-CD95 stimulation.
Moreover, our data demonstrate that the enzymatic activity of caspase 3 and caspase 8 (also known as
FLICE), both involved in CD95 signaling in human
chondrocytes (31), was down-modulated in IFN␥treated samples (Figures 5a and b). Caspase 3 is considered an executioner caspase and represents a pivotal
step of the CD95-dependent intracellular pathway. Upstream of its activation, CD95 engagement results in the
rapid recruitment of the adapter molecule FADD and
the caspase 8 proenzyme at the DISC, finally leading to
the activation of caspase 8, the most apical caspase.
It has been reported that treatment with the
protein synthesis inhibitor CHX inhibits FLIP expression within a few hours (37), thus supporting the evidence of a fast turnover rate of this protein; consistent
with these data, pretreatment of human OA chondrocytes with CHX at 10 ␮g/ml completely reversed the
effect of IFN␥ on caspase 8 proteolytic activity. This
finding supports the hypothesis that FLIP-enhanced
synthesis is involved in the survival signal provided by
IFN␥ in our model.
In conclusion, the observation that IFN␥ protects
chondrocytes against CD95-mediated apoptosis discloses a potential intriguing role for this cytokine in the
regulation of cartilage homeostasis. Based on our results, IFN␥ may contribute to creating an antiapoptotic
microenvironment in the cartilage under pathologic
conditions of the joint, such as OA, by counteracting the
effects of CD95L produced in the synovial fluids of these
patients. Our data reveal that IFN␥ exerts its antiapoptotic effect at relatively low concentrations, close to
those found in the synovial fluid of patients with rheumatic diseases (38,39). These findings may offer new
505
therapeutic tools to improve chondrocyte survival based
on the prevention of the abnormal increase of apoptosis
found in the cartilage after mechanical injury (40) or OA
disease. Moreover, the up-regulation of FLIP expression
and impairment of CD95 signaling demonstrated in our
experiments open new insights into the biologic effects
of IFN␥, which, at least in our model, constitute a
pivotal survival factor for human chondrocytes.
ACKNOWLEDGMENTS
The authors wish to thank Mrs. Graziella Salmi for
assistance with manuscript preparation and Mr. Luciano Pizzi
for technical assistance.
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expressions, associates, inhibition, osteoarthritis, apoptotic, flick, human, increase, inhibitors, protein, signaling, chondrocyte, interferon, cd95
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