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Introduction
Low back pain is a leading cause of morbidity. It is
estimated that about 70% of the population will experience low
back pain during their lives[1]. In recent years, intervertebral
disc (IVD) disorders and age-related degeneration have been
significant contributors to low back pain and spine-related
disability. There is great interest in understanding the
complex pathogenesis of IVD diseases and recent reports of the
existence of apoptotic cells in IVD have provided a new
insight into the pathophysiology of IVD
degeneration[2_8].
Fas/FasL system-mediated apoptosis is thought to play
an important role in the loss of disc cells that leads to
diminished generation, organization, and repair of the extracellular
matrix in the herniated lumbar disc tissues. The disc cells,
after herniation, undergo apoptosis via autocrine or paracrine
FasL mechanisms by the disc cells
themselves[2,5_8]. Fas (CD95) and Fas ligand (FasL) belong to the TNF family. The binding
of FasL with Fas triggers the formation of the
death-inducing signaling complex by recruiting an adaptor molecule
Fas-associated death domain (FADD) to the cytoplasmic tail of
Fas (C-terminal region). The subsequent autocatalytic
activation of a downstream cascade of caspases leads to the
cleavage of specific substrates and thus, the activation of
the apoptotic executioners[9_12].
Herniated IVD tissue has been shown to produce
pro-inflammatory cytokines, including matrix metalloproteinases
(MMPs), interleukin (IL)-1β, interleukin-1α, interleukin-6,
tumor necrosis factor-alpha (TNF-α), nitric oxide(NO), and
prostaglandin E2 (PGE-2)[13-18].
We proposed that normal disc cells can upregulate
apo-ptosis in an inflammation microenvironment attributed to the
production of a large number of inflammatory cytokines in
the disc degeneration process. Therefore, the goal of the
current study was to investigate the apoptotic effect and the
Fas gene expression on cultured rat IVD cells which were
stimulated by Fas ligand and IL-1β.
Materials and methods
Primary disc cell isolation All cell culture supplies were
purchased from Gibco BRL (Gaithersburg, MD, USA) unless
otherwise noted. Lumbar IVD (L3 to L6) from
Sprague_Dawley rats (aged 3 months, male, 455±29 g in weight) were
harvested immediately in a sterile environment after they
were killed. The nucleus pulposus (NP) was removed and
the inner annulus, including the transition zone (TZ), was
separated through an operating microscope. The
determination between the outer and inner annulus was based on
the amount of hydrated ground substance between the
lamellae. The outer annulus is a dense, fibrous tissue, with
little space between the oriented lamellar layers. The inner
annulus, including the TZ, contains more ground substance
which causes the lamellae to distend and become less
distinct and organized. For most discs, the outer one-third of
the annulus is designated as the outer annulus, and the
inner two-thirds are designated as the inner annulus (Figure 1).
The inner annulus fibrosus (IAF), including the TZ
between the annulus and the nucleus, was dissected and placed
in a humidified incubator with 5% CO2 at 37 ºC in Dulbecco's
modified Eagle's medium and Ham's F-12 medium
(DMEM/F-12) with 10% fetal bovine serum (FBS)
(Hyclone, Utah, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin
for 8 h. To isolate the cells, the disc tissues in
the DMEM/F-12 medium were digested with 0.25%
trypsin (including 0.02% EDTA) for 40 min followed by another treatment
with 0.1% collagenase for 8 h. After enzyme digestion,
the suspension was filtered through a 70 µm mesh. The
filtered cells were then washed with the DMEM/F-12
medium and a primary cell culture was started. About
1.5×106 cells were extracted from each rat lumbar disc. The rat
disc cells from the IAF including the TZ, were used for this
study.
Cell culture in selected concentrations of recombinant
rat IL-1β or the recombinant rat Fas ligand. When the
primary cell culture became confluent, the cells were
trypsinized and subcultured into 6-well plates at
3×105cells/well. The cells were cultured in DMEM/F-12 medium with
10% FBS, 100 U/mL penicillin, and 100 µg/mL streptomycin
in a humidified atmosphere with 5% CO2 at 37 ºC. When the
confluence in each well was over 80%, the medium was
replaced with medium containing 1% FBS without penicillin
and streptomycin. There were 5 treatment groups. In 3
treatment groups, the disc cells were cultured in the medium (1%
FBS) for 8 h. The medium was refreshed and 10 ng/mL
IL-1β (Cytolab/Peprotech Asia, Rehovot, Israel), 5 ng/mL FasL
(R&D Systems, Minneapolis, MN, USA), and 20 ng/mL FasL
was respectively added to the medium (1% FBS); the cells
were cultured for up to another 24 h. In the other 2 treatment
groups (10 ng/mL IL-1β+5 ng/mL FasL, and 10 ng/mL
IL-1β+20 ng/mL FasL), the cells were pretreated with 10 ng/mL
IL-1β for 8 h in the medium (1% FBS). The medium was then
refreshed and FasL (5, and 20 ng/mL) was respectively added
to the medium (1%FBS); the cells cultured for up to another
24 h. Cultures without addition of IL-1β or FasL acted as
the controls. After 32 h, the cells were double stained with
Annexin V_fluorescein isothiocyanate (Annexin V_FITC)
and propidium iodide (PI) (Bender MedSystems, Vienna,
Austria), and the cells were harvested for RNA extraction.
Flow cytometry(FCM) Apoptosis was determined by
staining cells with both Annexin V_FITC and PI, according
to the manufacturer's instructions. Annexin V_FITC is used
to quantitatively determine the percentage of cells
undergoing apoptosis. It relies on the property of cells to lose
membrane asymmetry in the early phase of apoptosis. In apoptotic
cells, the membrane phospholipid phosphatidylserine is
translocated from the inner leaflet of the plasma membrane
to the outer leaflet, thereby exposing phosphatidylserine to
the external environment. Cells that were positively stained
with Annexin V_FITC and negatively stained for PI were
considered apoptosis. Cells that were positively stained for
both Annexin V_FITC and PI were considered
necrosis[19,20]. To quantitate apoptosis, the cells were washed with cold
phosphate-buffered saline solution and then resuspended in
binding buffer (10 mmol/L HEPES
(N-2-hydroxyethylpipera-zine-N,-2-ethanesulphonic acid)/NaOH [pH 7.4], 140 mmol/L
NaCl, and 2.5 mmol/L CaCl2). The cells were stained with 5
µL Annexin V_FITC and 10 µL PI and then analyzed with
EpicsAltra (Beckman Coulter, CA, USA) FCM.
RT-PCR analysis of Fas gene transcription
Total RNA was isolated from the disc cells with TRIzol reagent
(Invitrogen, Carlsbad, CA, USA), according to the manufacture's directions. Single-strand cDNA templates
were prepared from 2 µg total RNA using oligo
(dT)18 and RevertAid M-MuLV reverse transcriptase (Fermentas,
Vilnius, Lithuania). Specific cDNA were then amplified by
PCR using the following primers (Sangon, Shanghai, China):
Fas sense primer: 5'-GCATCTTTGAGGGTTTGGA-3', antisense primer: 5'-CATTTGGTGTTGCTGGTTC-3' and
GAPDH sense primer: 5'-ACCACAGTCCATGCCATCAC-3', antisense primer: 5'-TCCACCACCCTGTTGCTGTA-3'.
PCR amplification (PCR System 2700, PE, CA, USA) from
cDNA was performed in a final volume of 50 µL containing
15 mmol/L MgCl2, 1.25 U Takara Taq, and 0.3
µmol/L specific primers (TaKaRa, Dalian, China). The cycling
conditions were: denaturation at 94 °C for 30 s, annealing (Fas
50 °C, GAPDH 58°C) for 30 s, and elongation at 72 °C for
60 s. The optimum cycle number was 30 cycles for Fas
and 25 cycles for GAPDH. All PCR products were
determined by 2% agarose gel electrophoresis by ethidium
bromide staining and visualized by UV transillumination. Gel
images were analyzed by densitometry using Scion Image
(Scion Corp, Frederick, MD, USA). Fas gene expression
data are presented as normalized to GAPDH expression.
Statistical analysis All experiments were performed at
least 3 times to ensure consistency. SPSS 11.0 software
(Chicago, IL, USA) was used for the statistics. Data were
compared using unpaired 2-tailed Student's
t-test analysis, with a P-value of 0.05 or less considered significant.
Results
Establishment of cultures in monolayer The primary
cells from the IAF, including the TZ of rat lumbar IVD,
became confluent after 9 d in the monolayer. Then the primary
cells were trypsinized and subcultured into 6-well plates with
3×105 cells/well. The first passage cells displayed a uniform,
rounded, chondrocyte-like morphology and achieved 80%
confluence after 7 d.
Evaluation of apoptosis When treated and cultured for
32 h, the apoptosis of the 6 groups was determined by
double staining with Annexin V_FITC and PI.
The apoptotic ratio of rat IAF and TZ cells were calculated as a percentage
of apoptotic cells/total cells (Table 1). Compared with the
control group, FasL (20 ng/mL), IL-1β (10 ng/mL)+FasL (5
ng/mL), and IL-1β (10 ng/mL)+FasL (20 ng/mL) induced
significant apoptosis of the disc cells (P<0.01). Apoptosis was
also induced by FasL 5 ng/mL (P<0.05); whereas, apoptosis
was not induced by IL-1β (10 ng/mL) (P>0.05).
IL-1β (10 ng/mL) enhanced the apoptosis-inducing effects
of FasL (5 ng/mL) and FasL (20 ng/mL) in disc cells (Figure 2,
P<0.01).
Transcription of Fas When treated and cultured for 32 h,
RNA was extracted from the monolayer-cultured rat disc cells.
RT-PCR was used for determining the transcription of the
Fas gene. The Fas gene was transcripted in the 5 treatment
and control groups (Figure 3). And the transcription levels
of the Fas gene in the 5 treatment groups were approximately
1.2_2.1-fold greater than the control group (respectively,
P<0.05). Additionally, group of IL-1β (10 ng/mL)+FasL (5
ng/mL) compared with group of FasL (5 ng/mL) and group of
IL-1β (10 ng/mL)+FasL (20 ng/mL) compared with group of FasL
(20 ng/mL) significantly upregulated transcription of Fas
(respectively, P<0.01).
Discussion
Fas is a membrane-bound receptor that is activated by
the binding of FasL and results in programmed cell
death/apoptosis. Park et al[6] reported the effect of Fas on disc
cells in human herniated disc tissues and found that the
percentage of Fas-positive cells correlated significantly with
patients' age, but not with the degree of disc degeneration
on magnetic resonance imaging. Wang et
al[8] also found that post-operative samples had an increased number of
Fas-positive cells in rat cervical degenerative disc models.
Anderson et al[21,22] detected a high expression of Fas following
both annular laceration in a rabbit model and fibronectin
fragment coculturing with rabbit IVD cells in
vitro. Fas is widely expressed in numerous different cell types
throughout the body, whereas Fas ligand expression appears to be
more restricted. The expression of Fas ligand in disc cells
could be detected in developing
embryos[23], degenerative
discs[7], normal discs[24], and scoliotic
discs[25]. Intervertebral discs with their extensive extracellular matrix are largely
avascular tissues and display anatomically isolation from the
hosts' immune system. Many studies have demonstrated
that Fas ligand should play a key role in the potential
molecular mechanism to maintain immune privilege of the
disc[7,24]. However, in degenerative
discs[7] and scoliotic
discs[25], Fas ligand had a close relationship with the apoptosis of disc
cells.
The inflammatory cytokine IL-1 plays an important role
in disc degeneration. IL-1 has been shown to increase the
synthesis of matrix-degrading enzymes (MMP-2, MMP-3,
MMP-13, and ADAMTS-4 (A Disintegrin-like and
metallo-protease with thrombospondin motifs 4) and to decrease the
synthesis of proteoglycan, collagen I and collagen II, and to
induce the expression of IL-6, cyclooxy-genase-2, stromolysin-1, and
PGE2[26_30]. IL-1β can also induce the
production of endogenous IL-1β by disc cells in
vitro. There is evidence to support that the positive feedback loop of
IL-1β exists in degenerative disc cells which upregulate the
production of mediators and thus can cause the cessation of
symptoms in intervertebral disc
herniation[30]. Our study demonstrated that FCM found no significant apoptosis after the
disc cells were treated with IL-1β (10 ng/mL) for 24 h, however,
the apoptotic rate could not deny the changes which
occurred to the disc cells. The effect of IL-1β on disc
degeneration has been unknown until now.
This study is the first to document normal disc cells
in vitro response to FasL with/without
IL-1β pre-treatment. Disc cells with IL-1β pre-treatment have a significant apoptotic
rate compared with control and disc cells without
IL-1β pre-treatment. This implies that the sensitivity of intervertebral
discs to FasL increased after IL-1β treatment, which led to a
high apoptotic rate at a low level of FasL in normal disc cells.
The present study, using RT-PCR, demonstrates that the
transcription of Fas in rat lumbar disc cells increased
significantly in the 5 treatment groups. It is important to
note that we detected the transcription of Fas on control
cells using RT-PCR. Park et
al[6] and Wang et
al[8] showed a similar result by means of immunohistochemistry, but
Anderson et al[22] reported no apparent transcription of Fas on
control discs based on the RT-PCR result.
There are 3 limits of the current study. One is that rat
disc cells only from the inner annulus fibrosus and TZ were
used, because rat NP primary cells could not proliferate and
disappeared after 3 weeks. The second limit was that our
study stimulated disc cells with IL-1β only for 24 h. It is
necessary to prolong the observation time to study whether
IL-1β can significantly induce apoptosis of disc cells. The
third limit was that we cultured the disc cells in the monolayer,
which can not completely represent the cells in
vivo, so it is necessary to culture disc cells in a 3-D culture system.
In conclusion, the results of this study showed that the
apoptotic rate of disc cells pretreated with IL-1β increased in
response to FasL in vitro and provided insights into
understanding the Fas/FasL system-mediated apoptosis in disc
cells which would be enhanced due to the inflammation
factor in degenerative discs.
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