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From the Department of Pharmacology,*
University
College, London; the Orthopedics Department,
Whittington Hospital; Novartis Institute for Medical
Sciences,
London; and the Department of
Immunology,§
Hammersmith Hospital, London,
United Kingdom
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Synovial membrane specimens were obtained from knee and hip joints from patients with RA undergoing joint replacement surgery. Control tissues were obtained from knee joints of patients undergoing amputation for sarcomata of the lower limb. The intimal surface of the synovial membrane was dissected, cut into small dices, and cells were dissociated through treatment with collagenase (2 mg/ml) (Worthington, Biochemical Corp., Lakewood, NJ) for 1 hour at 37°C. Dissociated tissue was sheared using a sterile syringe, filtered using a fine sterile gauze, and then washed and resuspended in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution (Gibco BRL, Paisley, UK) and kept in culture for 1 week as described by Croft et al.16 When confluent, cells were passaged using a trypsin-ethylenediaminetetraacetic acid solution. After the third passage the populations were on average 98% VCAM-1-positive and devoid (<1%) of monocyte or macrophage markers and therefore mainly consist of fibroblast-like synoviocytes (FLSs).
Immunocytochemistry
Cultured Cells
Cells were transferred to Permanox Lab-Tek chamber slides (Nunc) at a density of 2 x 104 cells/well and cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were fixed in methanol for 4 minutes followed by 1 minute in acetone, both kept at -20°C. After air-drying, the cells were washed twice in phosphate-buffered saline (PBS) and then incubated in 10% FBS/PBS for 20 minutes to saturate nonspecific binding sites. The cells were washed with PBS three times after each of the following steps. Hydrogen peroxide (3%) was applied for 5 minutes to quench endogenous peroxidase activity. All antibodies were diluted to their optimal concentration in 10% FBS/PBS. Anti-CD44v7/8 (clone VFF-17), anti-Ki67 (both from Serotec, Kidlington, UK), or anti-VCAM-1, clone BBIG-V1(4B2), (R&D Systems, Abingdon, UK) were applied to each well and incubated for 1 hour or overnight in the case of anti-Ki67. A negative control was performed by incubating cells with 10% FBS/PBS in the presence of mouse IgG1 antibodies (5 µg/ml; Sigma, Poole, UK). To visualize antibody binding, after three washes in PBS for 5 minutes, anti-mouse IgG biotin (Sigma) was added for 30 minutes, followed by avidin-peroxidase (Sigma) for 30 minutes, and the slides were stained using aminoethylcarbazole (AEC staining kit; Sigma) as substrate. The reaction was stopped in water. Except for the cells stained with anti-Ki67, nuclei were counterstained with Mayer’s hematoxylin for 5 minutes. The slides were mounted with Fluoromount-G and examined by light microscopy.
Synovial Membranes
The tissues were snap-frozen in liquid nitrogen and stored at -74°C until use. Ten-micrometer cryostat sections of these tissues were transferred to glass slides and allowed to air dry. The histology of the synovial lining layer was checked by staining the sections with 1% (w/v) toluidine blue in acetate buffer for 5 minutes. The slides were washed with running water, mounted with Fluoromount-G, left to dry, and examined by light microscopy using an Olympus PM-10AD. Fields of interest were photographed on Fujichrome T 64ASA film. For detection of CD44 splice variant expression, the sections were incubated with 10% (v/v) FBS in PBS to eliminate nonspecific background. They were next incubated for 1 hour at room temperature with mouse monoclonal anti-CD44v7/8 (clone VFF-17) antibodies diluted in PBS containing 10% FBS. A negative control was performed by incubating cells with 10% FBS/PBS in the presence of mouse IgG1 antibodies (5 µg/ml; Sigma). The sections were washed three times with PBS after each of the following steps. Anti-mouse IgG-fluorescein isothiocyanate was next applied and incubated for 30 minutes at room temperature. The sections were finally washed with water. The sections were mounted with Fluoromount-G and covered with glass coverslips, left to dry, and examined by a confocal microscope using a Leica TCS4D.
Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) and Southern Blotting
Total cellular RNA was isolated using Tri-reagent according to the
manufacturers instructions. Total RNA (2 µg) was reverse-transcribed
using a Ready to Go T-Primed First-Strand cDNA synthesis kit (Amersham
Pharmacia Biotech, Amersham, UK) for 60 minutes at 37°C. This kit
utilizes the Moloney murine leukemia virus reverse transcriptase and an
oligo (dT) primer to generate first strand cDNA. The completed
first-strand reaction mix was heated (5 minutes at 90°C) to
inactivate the reverse transcriptase and stored at -20°C.
Semiquantitive PCR was performed on cDNA (equivalent to 10 ng of RNA)
with oligonucleotide primers that were designed using primer-select
software (DNA Star) based on published sequences.7
In the
case of CD44, the primers are upstream and downstream of the variant
exon insertion point. CD44s: sense, CAG ACC TGC CCA ATG CCT TTG ATG GAC
C; antisense, CAA AGC CAA GGC CAA GAG GGA TGC C. For GAPDH the primers
are as follows: sense AAG GTG AAG GTC GGA GTC AAC; antisense GGC AGA
GAT GAT GAC CCT TTT GGC. [
-33P]dATP
was added to the reaction mixture to detect the PCR products formed.
The number of amplification cycles varied between 25 to 40, with each
cycle consisting of 30 seconds at 94°C, 30 seconds at 62°C
(annealing), and 30 seconds at 72°C. PCR products were
electrophoresed on a 6% polyacrylamide gel and radioactivity was
visualized using a phosphorimager (Fuji Bas 1000). For Southern
blotting, the PCR products, this time not labeled with
[33P]dATP, were separated on a 1.5% agarose
Tris borate-ethylenediaminetetraacetic acid gel, transferred
onto nylon membrane (Schleicher and Schuell, London, UK), and
hybridized with either CD44v7- or v8-specific probes in a Southern blot
protocol. 32P-labeled variant exon-specific
probes were generated from a variant CD44 template (containing variants
v3-10) using exon-specific primers and a Megaprime DNA labeling kit
(Amersham Pharmacia Biotech). The primers that were used are as
follows: CD44 v7 sense, GCC TCA GCT CAT ACC AAC; antisense, CCT TCT TCC
TGC TTG ATG ACC TCG TC CAT CCA ATG. CD44 v8 sense ATG GAC TCC AGT CAT
AGT ACA ACG C; antisense GTT GTC ATT GAA AGA GGT CCT GTC.
Western Blotting
Fibroblast-like synoviocytes from two patients with RA were cultured for 1 week after dissociation from the synovial membrane. The cells were trypsinized, counted (106 cells), and replated in 25-cm2flasks (Nunc, Life Technologies, Paisley, UK). The next day the cells were washed in PBS, solubilized in Laemmli sample buffer, and proteins were separated on a 10% (w/v) sodium dodecyl sulfate-acrylamide gel. After transferring protein on polyvinylidene difluoride Immobilon membrane (Millipore, Watford, UK), the blot was incubated overnight at 4°C with mouse monoclonal antibodies against CD44 (clone 5F12 CL4, NeoMarkers, New Market, UK) or against the variable exons CD44v7/8 (clone VFF-17, Serotech). A second, peroxidase-conjugated, antibody was applied for 1 hour at room temperature and antibody binding was visualized using enhanced chemiluminescence (Amersham Pharmacia Biotech).
Selection of CD44v7/8-Expressing Cells
Cells expressing CD44v7/8 were selected from a total population of 106 cells in a panning protocol using Dynabeads M-450 bound to goat anti-mouse IgG (Dynal, Bromborough, UK) according to the manufacturer’s protocol. Anti-CD44v7/8 was used at 5 µg/ml after extensive repeated washings in PBS supplemented with 1% (w/v) bovine serum albumin using Microcon-30 (Millipore) to remove azide. To remove antibody-coupled beads after selection, the cell suspension was treated with trypsin/ethylenediaminetetraacetic acid (Sigma) for 4 minutes after which FBS was added to a final concentration of 10%. Cells were washed and cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. Selection was assessed in an immunocytochemistry protocol before using the cells for further experimentation.
Cell Proliferation Measurements
Cells were plated in 24-well plates at 3 x 103 cells/well and left to adhere for 4 hours. Either CD44v7/8-enriched populations were compared with negatively selected cells (4 days of culture) or the effects of anti-CD44v3, anti-CD44v6, anti-CD44v7/8, or anti-VCAM-1 (10 µg/ml), was assessed in a total population (throughout a period of 11 days). The choice of the exon-specific antibodies in this assay is based on our evidence that the predominant splicing combinations in fibroblast-like synoviocytes from patients with RA are: CD44v3, CD44v6, and CD44v6-10. The variably spliced exons v4 or v5 have never been detected.16 For counting, cells were harvested and each cell suspension was resuspended in Isoton II (Coulter Electronics, Hialeah, FL). The cell suspension was then measured in Coulter counter type ZBI with a 100-µm orifice (Coulter Electronics). Cell numbers are expressed as cells per well or as percentage of control population.
3H-Thymidine Incorporation
One-week-old cells were seeded into 96-well plates at 2 x 103 cells/well in 100 µl of medium. Cells were left to adhere for 4 hours after which antibodies against CD44v3, CD44v6, CD44v7/8, or VCAM-1 at 10 µg/ml were added. Cultures were continued for another 3 days. 3H-thymidine (Amersham Pharmacia Biotech) was added at 0.5 µCi/well and left for 16 hours. This time period is appropriate for cells with a doubling time of around 60 hours (such as the fibroblast-like synoviocytes). Cells were then washed in PBS and fixed in 10% (w/v) trichloroacetic acid. DNA was solubilized in 0.25 mol/L NaOH for 1 hour and counted in the presence of UltimaGold (Packard Bioscience, Meriden, CT) in a scintillation counter (Beckman).
High-Density Oligonucleotide Array
Cells were incubated with or without dialyzed anti-CD44v7/8 (10
µg/ml) for 5 days. Cell numbers were counted, to check for growth
inhibition, and the cells were lysed in denaturing solution (ClonTech,
Basingstoke, UK) after which RNA was isolated and treated with DNase I
(ClonTech). cDNA was obtained with the use of Moloney murine leukemia
virus reverse transcriptase in the presence of
[-32P]dATP. The
32P-labeled cDNA was hybridized to a high-density
oligonucleotide array (Atlas Human Cell Cycle Array; ClonTech)
according to the manufacturer’s procedures. Radioactivity was
quantified and visualized using a phosphorimager (Molecular Dynamics
Storm II, Amersham Pharmacia Biotech).
Detection of Apoptosis
FLSs were cultured with or without dialysed anti-CD44v7/8 or anti-VCAM-1 mAbs. At day 5, cell supernatant was collected and the cells harvested by trypsinization. To record DNA histograms, the cells, together with the culture supernatant, were centrifuged (150 x g at room temperature for 5 minutes) and then washed once with PBS. They were fixed in cold 70% (v/v) ethanol in PBS and kept at 4°C until further processing. The cells were washed twice in phosphate-citrate buffer. RNase (100 µg/ml) was added to ensure that only DNA is stained. Propidium iodide (50 µg/ml) was subsequently added and the suspension analyzed for subG1 apoptotic cells using a flow cytometer (Becton Dickinson, Cowley, UK).
Statistical Analysis
All values given are the means ± SE. Statistical analyses were performed using analysis of variance or Student’s unpaired t-test. P values <0.05 were considered to be significant.
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Synovial sections were stained, using anti-CD44v7/8 antibodies,
and analyzed under the microscope. In tissues obtained from nondiseased
joints, no signal above background could be observed. Little staining
was detected in tissue treated with nonspecific mouse
IgG1-antibodies (negative control). Strong staining of the intimal
layer and deeper regions of the synovial membrane was visible in
tissues obtained from patients with RA (Figure 1)
. To test whether or not detection of
CD44v7/8 is a mere consequence of a general elevated expression of
CD44, we performed a semiquantitative RT-PCR experiment in which we
amplified the cDNA, obtained from freshly prepared synovial cells, at
various cycle numbers (Figure 2A)
. The
level of expression of standard-CD44 (CD44S) does not vary greatly
between cells derived from diseased and nondiseased joints. However,
high molecular weight CD44 splice variants (500 to 1,000 bp) are
exclusively present in cells derived from patients. When probed for the
presence of exon 7 and 8 in a Southern blot protocol, we confirmed that
these exons are indeed exclusively expressed in diseased joints (RA)
(Figure 2B)
. We next tested in a Western blotting protocol whether or
not the anti-CD44v7/8 specifically detects alternative splice-variants.
In these experiments we used cells derived from patients with RA. The
antibody directed against CD44 detects a whole range of isoforms
with molecular weights from Mr 85 kd to >200 kd, the variability being
due to different glycosylations and the presence of multiple
splicing combinations of CD44. The CD44v7/8 exon-specific antibody
detects a single protein of Mr 100 kd (Figure 2C)
. This finding is
entirely in agreement with our previous study where we found that the
CD44v7/8 epitope is most likely present in a single CD44v6-10 splicing
combination (see also 2B where there is only one PCR product (1.2 kb)
that contains both v7 and v8).16
From these findings we
conclude that the antibody detects a CD44 splice variant containing
exons v7/8 and that synovial membranes from arthritic joints, but not
from nondiseased joints, abundantly express this splice variant.
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Expression of CD44v7/8 is maintained in cultured cells obtained
from synovial tissues from RA patients but again could not be detected
from those obtained from nondiseased joints. Because the majority of
the cells also express VCAM-1 in these cultures, we identify them as
fibroblast-like synoviocytes (Figure 3A)
.
We do not, however, exclude that in the synovial tissue other cells
also express CD44 splice variants, this we are currently studying in
more detail. While assessing the percentage of cells expressing
CD44v7/8 in the cell culture dishes we observed, with time, an increase
in the percentage of cells bearing this epitope (Figure 3B)
. This
phenomenon was unlikely to be a consequence of a switch in CD44
splice-variant expression, for instance as a consequence of the culture
conditions, because, as mentioned earlier, we did not observe the
acquisition of these splice variants in cultures of cells obtained from
nondiseased joints.16
We therefore argued that the
enrichment of cells expressing the CD44v7/8 epitope could be a
consequence of a proliferative advantage.
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To address this matter we next studied co-expression of the splice
variants with the Ki67 epitope, a nuclear antigen present in
proliferating cells.17
We observed a high level of
co-expression between Ki67 and CD44v7/8 whereas a much lower
co-expression was observed in nonexpressing cells (Figure 4)
. This difference in co-expression was
significant and persisted in the presence of lower levels of fetal
bovine serum (down to 0.3%, v/v; data not shown). To further analyze
the relation between cell surface expression of CD44v7/8 and cell
proliferation, we selected fibroblast-like synoviocytes expressing this
epitope using a panning protocol with antibodies (anti-CD44v7/8)
coupled to magnetic beads. Using this method, with a 1-week-old
culture, we routinely obtained a nearly 100% CD44v7/8-positive
population (+ve) (Figure 5
, top). The
percentage of cells still expressing CD44v7/8 in the negatively
selected population (-ve) was estimated at 20%. Fibroblast-like
synoviocytes enriched for expression of CD44v7/8 have a higher
proliferation rate, we observed a 3.3-fold increase in cell number
after 4 days compared to a 2.6-fold increase of low expressers
(n = 4) (Figure 5
, bottom). From these data, we
calculate an average doubling time of 55 hours (96*Log[2]/Log[3.3])
for the positive population and an average doubling time of 69 hours
(96*Log[2]/Log[2.6]) for the negatively selected cells. This
difference in proliferation rates would predict an enrichment of
CD44v7/8-positive cells during culture which is slightly slower than
the one presented in Figure 3
. One should, however, bear in mind that
we did not entirely deplete the population of CD44v7/8-expressing cells
and these cells contribute to the total population proliferation rate.
These cell-doubling times are within the range published earlier using
similar culture times and conditions, in which it was shown that fast
proliferating fibroblasts had an average doubling time of 42 hours and
slow proliferating cells a doubling time of 72 hours.18
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To determine whether or not the expression of the CD44v7/8-epitope
plays a role in the regulation of the cell cycle, we next investigated
cell proliferation and DNA synthesis. For this purpose we took
fibroblast-like synoviocytes with a 1-week culture history and added
antibodies against the variable exons CD44v3, CD44v6, or CD44v7/8, or
antibodies against VCAM-1. The latter was included because the
fibroblast-like synoviocytes express very high levels of this adhesion
molecule19
for which no implication in proliferation had
been reported and could therefore serve as a negative control for
antibody treatment. After a culture period of 4 days, only the presence
of anti-CD44v7/8 antibodies reduced DNA synthesis and likewise after 11
days reduced cell proliferation (Figure 6)
. We interpret these findings as
indicating that expression of CD44v7/8 confers a proliferative
advantage on the fibroblast-like synoviocytes. To discern a molecular
mechanism, through which the antibodies against CD44v7/8 inhibit cell
proliferation, we used a high-density oligonucleotide array and
analyzed the profile of gene expression after treatment throughout a
5-day time period. In the antibody-treated cells we detected a
significant elevated expression of cyclin-dependent kinase
inhibitor p21Waf1/Cip120
and of growth arrest and DNA
damage-inducible protein GADD4521
and
GADD15322
(Figure 7
and
Table 1
). Because expression of GADD gene
products is associated with either growth arrest or programmed cell
death (apoptosis), we next analyzed the integrity of DNA at day 5 but
found no signs of fragmentation (data not shown). We take this finding
to mean that expression of these cell-cycle inhibitory genes, within
the time span of the experiment, is not linked to cell death.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The observation that antibodies against CD44v7/8 inhibit cell
proliferation is of interest for therapeutic reasons. First of all
there is evidence that hyperplasia of the fibroblast-like synoviocytes
plays a role in the development of a chronic erosive environment within
the synovial joint, most likely irrespective of the role of the immune
system therein.23,24
We base this idea on the findings
that 1) the degree of synovial hyperplasia correlates with the degree
of joint erosion,12,24
2) H2-c-fos transgenic
mice develop destructive arthritis,25
and 3) our finding
that fibroblast-like synoviocytes produce excessive amounts of
interleukin-6 independent of the presence of inflammatory mediators
such as tumor necrosis factor- and interleukin-1.26
High levels of interleukin-6 are instrumental in the reduction of
cartilage production27
and in its
breakdown.28
Secondly, in a recent study using a mouse
colitis model, it was shown that antibodies against the CD44v7 epitope
caused a full regeneration of the intestine.29
This effect
was explained by moderating an overshooting Th1 reaction. An excessive
Th1 response occurs in RA and is part of the chronic inflammatory
response.30
It remains the question, however, if
fibroblast-like synoviocytes in the synovial membrane are equally
sensitive to the blocking of the CD44v7/8 epitope. Results from
in situ studies have given good evidence for a contribution
of local proliferation of fibroblast-like synoviocytes to synovial
hyperplasia, both in humans15
and in experimental models
of arthritis.31
Given these considerations, the CD44v7/8
epitope could be an interesting target for pharmacological intervention
in the treatment of RA.
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Acknowledgements |
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Footnotes |
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Supported by the Thai Government and the Nuffield Foundation Oliver Bird Fund (to A. W. and D. C.). E. G. is a recipient of a Wellcome Career Development Award.
Accepted for publication August 21, 2000.
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1.2 kb) that contains
both v7 and v8. This autoradiograph is representative of three controls
(normal) and four
arthritic (RA) specimens
tested. C: Immunoblotting reveals the presence of a single
CD44 isoform (Mr 100 kd)
containing the v7/8 epitope. Cells obtained from two patients with RA
were lysed and subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis. Protein was transferred onto a polyvinylidene
difluoride membrane and probed with antibodies that recognize the core
protein of CD44 (CD44) or
those that recognize variable exons v7/8
(CD44v7/8). Antibody
binding was visualized after addition of a second,
peroxidase-conjugated anti-mouse IgG antibody in an enhanced
chemiluminescence protocol. Note the presence of numerous isoforms of
CD44 in patients with RA but only a single protein band that contains
both exons v7 and v8.
