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Induction of p16 at sites of cartilage invasion in the SCID mouse coimplantation model of rheumatoid arthritis.

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DOI 10.1002/art.11045
Induction of p16 at sites of cartilage invasion in the
SCID mouse coimplantation model of rheumatoid
arthritis
Rheumatoid arthritis (RA) is the most common type
of chronic inflammatory arthritis in humans. It is characterized
by synovial hyperplasia and pathologic immunologic phenom-
2069
ena mediating progressive joint destruction (1). There is
increasing evidence that activated RA synovial fibroblasts
(RASF) are key players in the process of joint destruction (2).
However, the mechanisms of activation of RASF are not
completely understood. The tumor suppressor protein p16
inhibits the binding of cyclin-dependent kinases 4 and 6 with
the D cyclins. Thereby, it blocks the phosphorylation of the
retinoblastoma (Rb) protein. Unphosphorylated Rb does not
release E2F and does not induce G1 entry into the cell cycle
(3). Therefore, p16 is a cell cycle inhibitor mediating G1 arrest.
Since it is encoded on human chromosome 9p in the
INK4a/ARF locus, p16 is also called p16INK4a. It is worth
noting that this genetic region is frequently mutated in patients
with cancer (4). Interestingly, Taniguchi et al demonstrated
that RASF were different from osteoarthritis synovial fibroblasts (OASF), normal synovial fibroblasts (NSF), and skin
fibroblasts in terms of regulation of the p16INK4a tumor
suppressor gene (5). Synthesis of p16INK4a was enhanced by
serum starvation, ␥-irradiation, and induction of senescence
and resulted in irreversible growth arrest. In contrast, expression of p16INK4a was not increased by these treatments, nor
was recovery of proliferation after addition of serumcontaining medium prevented in OASF or embryonic lung
fibroblasts. Intriguingly, administering p16 by adenoviral gene
transfer to rats with adjuvant-induced arthritis and to mice
with collagen-induced arthritis resulted in reduction of synovial cell hyperplasia and of mononuclear cell infiltration (5,6).
The role of the tumor suppressor protein p16INK4a in
RA is not well defined. Questions remain regarding the
expression pattern of p16INK4a in RA tissues, which cell types
express it, and the role of p16INK4a in joint destruction,
specifically at sites of cartilage invasion. Therefore, in the
present study we investigated the expression of p16INK4a in RA
synovial tissues and in control tissues by immunohistochemistry analysis. Levels of p16 messenger RNA (mRNA) and
protein in cultured RASF were investigated by real-time
polymerase chain reaction (PCR) and by immunofluorescence,
respectively. Finally, we explored whether RASF express
p16INK4a at sites of cartilage invasion in the SCID mouse
coimplantation model of RA.
Synovial tissue specimens were obtained from RA (n ⫽
13), OA (n ⫽ 2), and normal synovia (n ⫽ 1) at the time of
synovectomy, joint replacement, or trauma surgery. Samples
were fixed in 4% neutral buffered formalin for 6–8 hours,
dehydrated, and then embedded in paraffin using an automated tissue processor. All RA patients fulfilled the American
College of Rheumatology (formerly, the American Rheumatism Association) criteria for diagnosis of the disease (7).
Synovial fibroblasts were obtained by enzymatic digestion of synovial tissue from RA patients (n ⫽ 10) and normal
synovial tissue (n ⫽ 1) and cultured as previously described (8).
HeLa cells were used as p16-positive control cells. Cultured
synovial fibroblasts were detached with EDTA and stained
with anti–HLA–DR (BD PharMingen, Basel, Switzerland) and
anti–Thy-1 (clone ASO2; Dianova, Hamburg, Germany). Cells
were analyzed on a FACSCalibur flow cytometer and data
were processed using CellQuest software (Becton Dickinson,
San Jose, CA).
For immunohistochemistry analysis, tissue sections
were dewaxed in xylol and rehydrated in decreasing concen-
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Figure 1. Expression of p16 by immunohistochemistry. Only limited p16 expression occurred in osteoarthritis
(OA) synovium, but strong expression was observed in uterus glands (positive control). Expression of p16 was
detected in the synovial sublining of rheumatoid arthritis patients (RA1) as well as in the lining layer in these
patients (RA2) (original magnification ⫻ 100 for OA; ⫻ 200 for RA1; ⫻ 400 for uterus and RA2).
trations of ethanol, pretreated by microwave heating in 0.01M
citrate buffer (pH 6.0), and kept at 70°C for 30 minutes in a
heat incubator for antigen retrieval. Slides were blocked at
room temperature with blocking solutions A and B (Vector,
Burlingame, CA), and afterwards with 2% horse serum in 4%
nonfat milk/Tris HCl (0.1M, pH 7.6). Primary anti-p16 antibodies (clone G175-1239; diluted 1:100, final concentration 5
␮g/ml) (BD PharMingen) were applied at 4°C overnight.
Mouse isotype-matched IgG sera were used as negative controls. The streptavidin–biotin detection system was used for
amplification of the signal, and nitroblue tetrazolium/BCIP
(Roche, Rotkreuz, Switzerland) served as color reagent. Tissue
sections from normal uterus and pancreas were used as
positive controls. For each patient, the percentage of positive
cells was evaluated on at least 10 high-power fields (400⫻
magnification). All immunohistochemical reactions were repeated at least twice.
To determine the cell types expressing p16, parallel
sections of synovial tissue specimens were stained with antibodies against CD68 (dilution 1:60, final concentration 6
␮g/ml) (Dako, Glostrup, Denmark). Specimens from SCID
mice were analyzed for the proliferation marker Ki67 with
anti-Ki67 antibodies (dilution 1:100, final concentration 1
␮g/ml) (Dako). Incubation was performed overnight at 4°C. To
amplify the signal, for the anti-CD68 and anti-Ki67 antibodies,
the same streptavidin–biotin detection system was applied. In
addition, SCID mouse sections were investigated with rabbit
anti-active caspase 3 antibodies (dilution 1:100, final concentration 0.5 ␮g/ml) (Cell Signaling Technology [Frankfurt,
Germany], distributed by Bioconcept, [Allschwil, Switzerland])
using microwave pretreatment, the streptavidin–biotin detection system, and diaminobenzidine as color reagent (Vector).
Tissue sections from juvenile thymus, breast carcinoma, and a
neurologic tumor were used as positive controls.
Immunofluorescence was also used for detection of
p16INK4a. RASF (n ⫽ 10) or NSF (n ⫽ 1) from passages 3–7,
as well as HeLa cells, were cultured on chamber slides (Labtec
II; Nunc, Basel, Switzerland) and fixed with methanol/acetone
(1:1) for 10 minutes at ⫺20°C. After rehydration in buffer
(0.1M Tris HCl [pH 7.6]), slides were blocked with 2% horse
serum in 4% nonfat milk. Slides were incubated for 30 minutes
with the above-mentioned anti-p16 antibodies (dilution 1:500,
final concentration 1 ␮g/ml). Mouse isotype-matched IgG
served as a negative control. For detection, Cy3-conjugated
sheep anti-mouse serum (dilution 1:1,000, final concentration
0.14 ␮g/ml) (Dianova, Hamburg, Germany) was applied for 30
minutes. After mounting, slides were analyzed with a Zeiss
immunofluorescence microscope and positive cells were
counted.
For quantification of p16 mRNA by real-time PCR,
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2071
Figure 2. A, Expression of p16 in the SCID mouse coimplantation
model of rheumatoid arthritis (RA). Limited cartilage invasion of
normal synovial fibroblasts (NSF) and only few p16-positive cells were
detected. In RASF, p16 was induced at sites of cartilage invasion, since
in vitro almost all of the same cells were p16 negative (magnification
⫻400). B, Quantification of p16-positive cells at sites of cartilage
invasion. Values are the mean and SEM. The expression of p16 was
significantly higher in RASF than in NSF (P ⬍ 0.05 by Mann-Whitney
U test).
RNA was extracted from RASF (n ⫽ 8), NSF (n ⫽ 1), and
HeLa cells using the RNA Miniprep Kit (Stratagene Europe,
Amsterdam, The Netherlands), with DNase treatment. After
reverse transcription (RT) (MultiScribe; PE Applied Biosys-
tems, Rotkreuz, Switzerland), the generated complementary
DNA was amplified by quantitative real-time PCR using
specific primers and a TAMRA/FAM label. Primers and probe
for p16 were checked for specificity by GenBank analysis.
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Their sequences were as follows: forward primer 5⬘-CCAACG-CAC-CGA-ATA-GTT-ACG-3⬘, reverse primer 5⬘GGG-CGC-TGC-CCA-TCA-3⬘, probe 5⬘-CAT-GAC-CTGGAT-CGG-CCT-CCG-A-3⬘. Expression of 18S ribosomal
RNA using predeveloped primer/probes (PE Applied Biosystems) served as internal standard. DNA contamination was
evaluated by using the mRNA sample (non-RT control) as
reaction template.
SCID mice were obtained from Charles River (Sulzfeld, Germany) and kept permanently under sterile conditions.
Implantation of RASF (n ⫽ 5 patients, 20 animals) and NSF
(n ⫽ 1 patient, 9 animals) together with normal human
cartilage was performed as previously described (9). After 60
days, mice were killed, and the implants fixed in 4% buffered
formalin and embedded in paraffin according to standard
procedures. Paraffin-embedded sections were stained with
hematoxylin and eosin or analyzed for expression of p16 by
immunohistochemistry. Invasion into cartilage was quantified
according to a semiquantitative score ranging from 0 to 4,
referring to the number of invading cells and the number of
affected cartilage sites.
For statistical analysis, the Mann-Whitney U test was
used. P values less than 0.05 were considered significant. Data
were expressed as the mean ⫾ SEM.
In the experiments using immunohistochemistry and
specific anti-p16 antibodies, p16INK4a was found to be expressed in the synovial lining as well as in the sublining layer of
RA synovial tissues (Figure 1). No p16INK4a staining occurred
in normal synovium (results not shown), and there was only
limited expression in OA synovial tissues. A mean ⫾ SEM of
6.5 ⫾ 2.0% of all cells in RA synovial tissue expressed
p16INK4a, and this expression was significantly increased when
compared with OA and normal synovial tissues (combined
mean ⫾ SEM 0.25 ⫾ 0.25%; P ⫽ 0.014). Moreover, in a
subgroup of patients with RA (3 of 13), higher p16INK4a
expression, ranging from 14% to 23%, was detected. However,
the staining patterns of p16 and CD68 were different. In some
regions CD68 and p16 colocalized, whereas in others CD68negative areas were clearly positive for p16, indicating that
CD68-negative fibroblast-like cells also expressed p16.
In vitro, ⬎98% of the investigated synovial cells were
positive for the fibroblast marker Thy-1, and ⬍1% stained
positive for type II major histocompatibility complex molecules. As determined by immunofluorescence, the constitutive
expression of p16INK4a protein was 6 ⫾ 0.5% positive cells in
untreated RASF and 4% in NSF, whereas all HeLa cells
exhibited staining for p16INK4a (results not shown). Accordingly, the levels of expression of p16INK4a mRNA as quantified
by real-time PCR were 100 times lower in RASF than in HeLa
cells.
Cultured RASF from 5 patients, showing a mean level
of 6% p16-positive cells, as well as NSF from 1 patient were
engrafted under the renal capsule of SCID mice. In accordance
with our previous findings (9), RASF invaded into the coimplanted cartilage significantly more strongly than did NSF
(mean ⫾ SEM invasion score 3.0 ⫾ 0.18 for RASF, 1.2 ⫾ 0.18
for NSF; P ⬍ 0.01). Most intriguingly, RASF expressed the
tumor suppressor protein p16 in 40% of the cells at sites of
cartilage invasion (Figure 2A). Implanted NSF demonstrated
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only minimal cartilage invasion, and ⬍2% of the cells adjacent
to the cartilage were p16 positive (Figure 2A). In addition,
parallel SCID mouse sections were immunohistochemically
stained for the proliferation marker Ki67 and for the cleaved
fragment of caspase 3, indicating apoptosis. In both staining
reactions, only negligible signals were detected (results not
shown), suggesting neither proliferation nor the occurrence of
apoptosis. Of note, control tissues were positive for the determined markers.
The present study reveals that p16 is induced in vivo in
RASF at sites of cartilage invasion in the SCID mouse
coimplantation model of RA. The in vivo induction mechanism
is as yet unclear, but in vitro, Taniguchi et al (5) demonstrated
induction of p16 in RASF by ionizing irradiation, low serum
concentration, or sensescence. They proposed a novel treatment of RA, since in rat adjuvant arthritis and mouse collageninduced arthritis they could reduce synovial hyperplasia and
mononuclear cell infiltration by gene transfer of p16 (5,6).
Herein we report that the cartilage invasion process
appears to be associated with the expression of p16. We
suggest that p16 could mediate arrest of the cell cycle at sites
of cartilage invasion, since the cells were also negative for the
proliferation marker Ki67. This notion is supported by the
results of investigations by Jung et al (10) demonstrating low
proliferation in human colorectal adenocarcinomas at sites of
tumor invasion, in association with the expression of p16.
Furthermore, in tumor cells the transfection of p16 also
resulted in apoptosis (11). However, in our in vivo study the
expression of p16 at sites of invasion was not correlated with
the detection of apoptotic cells. This result is in accordance
with the findings of other studies (5) demonstrating that in
vitro p16 gene transfer of RASF inhibited only the cell cycle,
but did not effect apoptosis. The finding that p16 is expressed
in RA tissues is in contrast with the results of Taniguchi et al
(5), who reported no p16 expression in 3 RA tissues, studied
using Western blot analysis. However, in the present study,
immunohistochemistry analysis clearly showed positive staining for p16 in ⬃6% of the cells. Therefore, we hypothesize that
in the study by Taniguchi and colleagues, the 3 RA samples
investigated might not be representative for the expression of
p16 in all subsets of RA tissues, at least in samples derived
from Europe.
In summary, we have shown that p16 was induced at
sites of cartilage invasion, most likely mediating cell cycle
arrest, but not apoptosis. From these data we propose a role
for the tumor suppressor protein p16 in RA, specifically in the
process of cartilage invasion. We assume that p16-expressing
and low-proliferating RASF, which have been shown previously to be positive for the apoptosis inhibitor sentrin (12) and
negative for the tumor suppressor PTEN (13) and to strongly
express matrix-degrading enzymes (14), represent a phenotype
associated with the arrest of the cell cycle and thereby maintain
the invasive phenotype of cells mediating joint destruction in
RA.
We thank Maria Comazzi and Ferenc Pataky for their excellent
technical assistance, and Diego Kyburz and Janine Rethage for help in the
FACS analysis. Drs. Kuchen and R̂ihoŝková’s work was supported by the
EMDO Foundation. Dr. Seemayer’s work was supported by the Swiss
National Science Foundation and the EMDO Foundation.
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2073
Peter Kuenzler, MSc
Stefan Kuchen, MD
Veronika Řihošková, MSc
Beat A. Michel, MD
Renate E. Gay, MD
Michel Neidhart, PhD
Steffen Gay, MD
University Hospital Center of
Experimental Rheumatology
Zurich, Switzerland
Christian A. Seemayer, MD
University Hospital Center of
Experimental Rheumatology
Zurich, Switzerland
and University Hospital
Institut of Pathology
Basel, Switzerland
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invasion, site, induction, mode, p16, mouse, arthritis, cartilage, rheumatoid, scid, coimplantation
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