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Modulation of RANKL and osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis in response to disease-modifying antirheumatic drug treatment and correlation with radiologic outcome.

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Arthritis & Rheumatism (Arthritis Care & Research)
Vol. 59, No. 7, July 15, 2008, pp 911–920
DOI 10.1002/art.23818
© 2008, American College of Rheumatology
ORIGINAL ARTICLE
Modulation of RANKL and Osteoprotegerin
Expression in Synovial Tissue From Patients With
Rheumatoid Arthritis in Response to DiseaseModifying Antirheumatic Drug Treatment and
Correlation With Radiologic Outcome
DAVID HAYNES,1 TANIA CROTTI,1 HELEN WEEDON,2 JOHN SLAVOTINEK,3 VIRGINIA AU,3
MARK COLEMAN,3 PETER J. ROBERTS-THOMSON,3 MICHAEL AHERN,2 AND MALCOLM D. SMITH4
Objective. To demonstrate the effect of treatment with disease-modifying agents on the expression of osteoprotegerin
(OPG) and RANKL in the synovial tissue from rheumatoid arthritis (RA) patients and to correlate these changes with
radiologic damage measured on sequential radiographs of the hands and feet.
Methods. Synovial biopsy specimens were obtained at arthroscopy from 25 patients with active RA (16 of whom had a
disease duration <12 months) before and at 3– 6-month intervals after starting treatment with a disease-modifying agent.
Immunohistologic analysis was performed using monoclonal antibodies to detect OPG and RANKL expression, with
staining quantitated using computer-assisted image analysis and semiquantitative analysis techniques. Serial radiographs of the hands and feet were analyzed independently by 2 radiologists and a rheumatologist using the van der Heide
modification of the Sharp scoring method.
Results. Thirteen patients achieved a low disease state as defined by a disease activity score <2.6 while 19 patients
achieved an American College of Rheumatology response >20% after disease-modifying antirheumatic drug (DMARD)
treatment. Successful DMARD treatment resulted in an increase in OPG expression and a decrease in RANKL expression
at the synovial tissue level, which correlated with a reduction in erosion scores measured on annual radiographs of the
hands and feet.
Conclusion. Successful treatment-induced modulation of OPG and RANKL expression at the synovial tissue level,
resulting in a reduction in the RANKL:OPG ratio, is likely to have a significant impact on osteoclast formation and joint
damage in patients with active RA.
Rheumatoid arthritis (RA) is characterized by inflammation of the synovial membrane leading to invasion of synovial tissue into the adjacent cartilage matrix with degradation of articular cartilage and bone as a consequence.
This results in erosion of bone, which is often observed as
marginal joint erosions radiographically and is predictive
of a poorer prognosis (1). Although the pathophysiologic
mechanisms for cartilage and bone destruction in RA are
not yet completely understood, it is known that matrix
metalloproteinases, cathepsins, and mast cell proteinases
can contribute to cartilage and bone destruction in RA
(2– 4). However, it is now clear that osteoclast formation
Supported by grants from the National Health and Medical Research Council of Australia, the Clive and Vera Ramaciotti Foundation, the J. H. and J. D. Gunn Foundation,
and the Rebecca L. Cooper Medical Research Foundation.
1
David Haynes, PhD, Tania Crotti, BHlthSci, PhD: University of Adelaide, Adelaide, South Australia, Australia;
2
Helen Weedon, BSc, Michael Ahern, MBBS, PhD, FRACP:
Repatriation General Hospital, Daw Park, Adelaide, South
Australia, Australia; 3John Slavotinek, MBBS, FRANZCR,
Virginia Au, MBBS, FACR, FRANZCR, Mark Coleman,
MBBS, FRCPA, Peter J. Roberts-Thomson, DPhil(Oxon),
FRACP: Flinders Medical Centre, Adelaide, South Australia, Australia; 4Malcolm D. Smith, MBBS(Hons), PhD,
FRACP: Flinders Medical Centre and Repatriation General
Hospital, Adelaide, South Australia, Australia.
Address correspondence to Malcolm D. Smith,
MBBS(Hons), PhD, FRACP, Rheumatology Research Unit,
Repatriation General Hospital, Daws Road, Daw Park,
South Australia, 5041 Australia. E-mail: malcolm.smith@rgh.
sa.gov.au.
Submitted for publication September 18, 2007; accepted
in revised form February 19, 2008.
INTRODUCTION
911
912
and activation at the cartilage–pannus junction is an essential step in the destruction of bone matrix in patients
with RA (5– 8). A number of inflammatory cytokines found
in the synovial tissue of patients with RA (interleukin-1␣
[IL-1␣] and IL-1␤, IL-6, tumor necrosis factor ␣, macrophage colony-stimulating factor [M-CSF]) have the potential to promote osteoclast formation and bone resorption
(9 –11). However, recent evidence indicates that the interaction between RANKL and RANK has an essential role in
osteoclastogenesis (5,6,12,13). RANKL is expressed on osteoblasts, fibroblasts, and T cells, whereas RANK is mainly
expressed on preosteoclasts, possibly of macrophage lineage. There is a naturally occurring inhibitor of the
RANKL interaction with RANK, called osteoprotegerin
(OPG), which binds RANKL with high affinity, preventing
RANKL from interacting with RANK (14).
We have previously published reports on the expression
of both RANKL (15) and OPG (16) in the synovial tissue of
patients with inflammatory arthritis and osteoarthritis as
well as normal synovial tissue (17) and demonstrated a
lack of OPG expression with significant RANKL expression in the synovial tissue from patients with active RA.
We hypothesized that successful treatment with diseasemodifying antirheumatic drugs (DMARDs) would reduce
RANKL expression and increase OPG expression, altering
the RANKL:OPG ratio and suppressing osteoclast formation in the synovial tissue of patients with RA. The goal of
the present study was to test this hypothesis in a cohort of
RA patients with active disease initiating DMARD treatment and to attempt to correlate the changes in synovial
tissue expression of RANKL and OPG with radiologic outcomes in this patient cohort.
PATIENTS AND METHODS
Patients. Twenty-five RA patients with active synovitis,
including an involved knee joint, were recruited for the
study. All RA patients fulfilled the American College of
Rheumatology (ACR; formerly the American Rheumatism
Association) criteria for RA (18). The mean age of the
patient group was 68.4 years (range 47– 87 years) with 14
men and 11 women. Eighteen of the 25 patients were
seropositive for rheumatoid factor, while 2 of the 7 seronegative patients had radiologic evidence of erosions at
study entry. The mean disease duration was 4.3 years
(range 0.1–24 years); 16 patients had a disease duration ⬍1
year. Decisions about which DMARD to use to treat an
individual patient were made by the treating rheumatologist and were not influenced by participation in this study.
DMARDs used included methotrexate (6 patients), intramuscular (IM) gold (6 patients), methotrexate and IM gold
(9 patients), sulfasalazine (2 patients), cyclosporin A (1
patient), and hydroxychloroquine (1 patient). No anti–
tumor necrosis factor (anti-TNF) agents were used to treat
this patient group because the study preceded the general
availability of these therapeutic agents in Australia. Failure to respond to treatment with the original DMARD
along with clear evidence of radiologic progression led to
withdrawal from the study. All patients were followed up
at regular intervals (3– 6 months) for clinical (tender and
Haynes et al
swollen joint counts, visual analog scales for pain, patient
and physician global assessments, and a modified Health
Assessment Questionnaire [HAQ]), laboratory (C-reactive
protein, erythrocyte sedimentation rate, and rheumatoid
factor), and radiologic (radiographs of the hands and feet
taken annually) parameters. Response to DMARD treatment was assessed by calculating a disease activity score
in 28 joints (DAS28) (19) and ACR response (20). All
patients gave informed consent, and the study protocol
was approved by the research and ethics committee of the
Repatriation General Hospital.
Synovial tissue. A small-bore arthroscopy (2.7-mm arthroscope; Dyonics, Andover, MA) was performed with
patients under local anesthesia as previously described
(21) at baseline and at 3, 6, 12, 18, 24, and 36 months after
starting DMARD treatment. Biopsy specimens of synovial
tissue were obtained from all accessible regions of the knee
joint, but mainly from the suprapatellar pouch. The samples were separately snap-frozen in Tissue-Tek OCT
(Miles Diagnostics, Elkhart, IN) and stored at ⫺80°C until
used. Cryostat sections (6 ␮m) were mounted on glass
slides (Superior Marienfeld, Baden-Wurttemberg, Germany). The glass slides were boxed and stored at ⫺20°C
until immunohistologic analysis.
Immunohistochemistry. Serial sections were stained
with the following mouse monoclonal antibodies (mAb):
anti-human OPG antibodies (mAb 805 and mAb 8051;
R&D Systems, Minneapolis, MN); anti-human TRANCE
(mAb 626; R&D Systems); anti-CD68 (clone EBM11; Dako
Australia, Botany Bay, New South Wales, Australia) to
detect macrophages; mAb 67 (Serotec, Kidlington, Oxford,
UK), which recognizes CD55, to detect fibroblast-like synoviocytes; anti-CD3 (BD Biosciences, San Jose, CA) and
anti-CD45RO to detect T cells and memory T cells, respectively; anti-CD22 (Central Laboratory of the Netherlands
Red Cross Blood Transfusion Service, Amsterdam, The
Netherlands) to detect B cells; anti-CD38 (BD Biosciences)
to detect plasma cells; and anti– granzyme B (Novo Castra
Laboratories, Newcastle upon Tyne, UK). Endogenous peroxidase activity was inhibited using 0.1% sodium azide
and 1% hydrogen peroxide in Tris–phosphate buffered
saline. Staining for cell markers was performed as described previously (15–17). Following a primary step of
incubation with mAb, bound antibody was detected according to a 3-step immunoperoxidase method. Horseradish peroxidase (HRP) activity was detected using hydrogen
peroxide as substrate and aminoethylcarbazole (AEC) as
dye. Slides were counterstained briefly with hematoxylin
solution and mounted in Gurr Aquamount (BDH, Poole,
UK). Affinity-purified HRP-conjugated goat anti-mouse
antibody was obtained from Dako, affinity-purified HRPconjugated swine anti-goat Ig from Tago (Burlingame, CA),
and AEC from Sigma (St. Louis, MO). The specificity of the
antibodies against RANKL (15) and OPG (16) has previously been demonstrated by absorption studies using purified RANKL and OPG.
RANKL and OPG in RA Synovial Tissue
913
Quantitation of immunohistochemical labeling. After
immunohistochemical staining, sections stained for OPG
(mAb 805 and 8051) and RANKL were analyzed in a random order by computer-assisted image analysis, analyzing
6 high-power fields for each section as previously described (22,23). In addition, these sections were scored by
a semiquantitative method on a 5-point scale by 2 independent observers in a random order, as described previously (15,16,24).
determined, defined as the mean cycle at which the fluorescence curve reached an arbitrary threshold. The ⌬Ct for
each sample was then calculated according to the formula
Ct target gene ⫺ Ct hARP; ⌬⌬Ct values were then obtained
by subtracting the ⌬Ct of a reference sample (the average
⌬Ct for osteoarthritis samples) from the ⌬Ct of the studied
samples. Finally, the levels of expression of the target
genes in the studied samples as compared with the reference sample were calculated as 2⫺⌬⌬Ct.
Real-time polymerase chain reaction on synovial tissue. Complementary DNA (cDNA) samples were prepared
from synovial biopsy specimens from 5 RA patients using
the initial synovial tissue obtained before starting DMARD
treatment and again using the biopsy specimens obtained
when there was significant improvement in disease activity as defined by the DAS28. As controls for these patients,
cDNA was prepared from synovial tissue obtained at arthroscopic biopsy in 5 patients with osteoarthritis of the
knee joint. Real-time polymerase chain reaction (PCR) was
performed using Platinum SYBR Green quantitative PCR
Supermix-UDG (Invitrogen Life Technologies, San Diego,
CA) as per the manufacturer’s recommendations. Amplification was carried out in a Rotor-Gene 3000 (Corbett Life
Science, Mortlake, New South Wales, Australia) with
SYBR Green detection and melt curve analysis. Oligonucleotide primers that were used have been described previously, and are specific for OPG and RANKL (25). The
endogenous reference gene hARP (26) was used to normalize threshold cycle (Ct) data obtained from the genes investigated. Reaction mixtures contained 1 ␮l of 1:5 diluted
cDNA, 7.5 ␮l Platinum SYBR Green quantitative PCR Supermix-UDG, 300 nM each of forward and reverse primer,
and diethyl pyrocarbonate–treated water to a final volume
of 15 ␮l. All samples were investigated in triplicate and
the melting curves obtained after each PCR amplification
confirmed the specificity of the SYBR Green assays. Optimization of forward and reverse primer concentrations
between 50 nM and 900 nM was evaluated to obtain the
combination of primers with the lowest Ct value. For each
target gene (OPG and RANKL) and endogenous reference
gene (hARP), a concentration of 300 nM of both forward
and reverse primers yielded the lowest Ct values, with the
highest increase in fluorescence. Validation experiments
were performed to demonstrate that amplification efficiencies of the target genes and the endogenous reference gene
were approximately equal. Complementary DNA was prepared from pooled RNA samples and diluted in a 2-fold
dilution series over 6 orders of magnitude. Target and
reference genes were then amplified in separate tubes using this cDNA dilution series and Ct were values obtained.
The difference in Ct (⌬Ct) for each sample was then calculated as outlined below and data were plotted against the
log cDNA dilution. If the absolute value of the slope is
⬍0.1, the efficiencies of the target and reference genes are
similar, and the ⌬⌬Ct calculation for the relative quantification of target may be used (27). The slope for OPG hARP
was 0.078 and for RANKL hARP was 0.0125. Relative
expression of the target genes in the studied samples was
obtained using the difference in the Ct (⌬⌬Ct) method.
Briefly, for each sample, a value for threshold (Ct) was
Grading of serial radiographs. Radiographs of the
hands and feet were obtained for all patients included in
this study ⬃12 months apart for the duration of the study
using a standard technique. The radiographs were graded
by 2 radiologists and a rheumatologist, using the van der
Heide modification of the Sharp technique (28), after first
standardizing against each other using a series of hand and
feet radiographs from 4 RA patients not included in this
study. The radiographs were graded separately by the 3
assessors without knowledge of the clinical outcome, but
with knowledge of the timing of the radiographs. The
scoring system results in a maximum score of 210 for joint
space narrowing and 280 for erosions. A disagreement
between scorers of up to 5% of the maximum score was
allowed, with the final score being an average of the 3
scores. Any disagreement above this level was settled by
consensus, with all 3 assessors scoring all radiographs for
a single patient together in the same session. This was
necessary for 4 of the 25 patients included in this study.
Statistical analysis. Results are presented as the
mean ⫾ SD. Tests for normality were applied to the data.
Semiquantitative scores were treated as nonparametric
data. Data generated by computer-assisted digital image
analysis (integrated optical density [IOD]) were generally
normally distributed. Changes within the groups were analyzed using Wilcoxon’s signed rank test for data that were
not normally distributed (semiquantitative scores) and
Student’s paired t-test for normally distributed continuous
data (IOD). Separate linear regressions of each parameter
versus time in months were performed. To account for the
correlation between repeated observations on the same
patient in the analyses, generalized estimating equations,
assuming an exchangeable correlation structure, were
used in the regressions. The analyses were also carried out
for the differenced data, in which the first synovial biopsy
result was subtracted from each subsequent synovial biopsy result for each of the variables.
RESULTS
Clinical and demographic features. All patients included in this study had active RA, with a mean tender
joint count of 17.7 (range 5–26), mean swollen joint count
of 13.8 (range 3–27), mean modified HAQ score of 2.3
(range 1.3–3.0), and mean DAS28 of 5.9 (range 4.4 –7).
There was a significant decrease in DAS28 with treatment
(P ⫽ 0.000). Thirteen patients attained a low disease activity state as defined by a DAS28 score ⬍2.6 following
914
Haynes et al
DMARD treatment. Nineteen patients attained a significant
ACR response to treatment, defined as a ⬎20% ACR response to treatment, but 6 of these patients did not achieve
a low disease activity score as defined by a DAS28 ⬍2.6.
Radiologic outcomes for the patient group. Despite the
predominantly short disease duration of this patient
group, patients had evidence of erosions (mean erosion
score 16.2, range 0 –211) and joint damage (mean joint
space narrowing 16.2, range 0 – 69) at the time of study
entry. The patients who failed to respond to DMARD treatment had significant increases in joint damage over time,
whereas the patients who responded to DMARD treatment
had no real change in radiologic damage over time (Figure
1). Patients who were clearly not responding to treatment
and had radiographic evidence of progressive joint damage
were removed from the study and offered alternative treatment. For this reason, there was a longer radiologic followup in the responder group than the nonresponder
group (7 years versus 3 years).
Immunohistochemistry results. As previously described (16), 2 distinct patterns of staining for OPG in
synovial tissue were seen: mAb 805 stained exclusively
endothelial cells, while mAb 8051 stained mainly the lining layer of the synovial membrane and weakly stained
endothelial cells. As shown in Tables 1 and 2, the expression of OPG in both blood vessels (Figure 2) and synovial
lining (Figure 3) was either low or absent in patients with
active RA, whereas the expression of OPG increased as a
result of response to DMARD treatment (P ⫽ 0.005). In
contrast, RANKL expression was high in the synovial tissue of patients with active RA (Figure 4) and decreased as
a result of successful DMARD treatment (P ⫽ 0.003). The
major changes in synovial tissue inflammatory infiltrate
were reductions in macrophage and T cell content as a
result of DMARD treatment, with little change in B lymphocytes, plasma cells, or granzyme B–positive cells, as
previously shown (29,30). We assessed whether the
change in T cell content of synovial tissue from patients
treated with DMARDs could explain the decrease in
RANKL expression in these patients by fitting regression
models using generalized estimating equations that included time and CD3 content as covariates with an exchangeable correlation structure. The effect of DMARD
treatment on CD3 content of synovial tissue did not explain the changes seen in RANKL content of synovial
tissue from patients treated with DMARDs. There was an
increase in fibroblast lining cell (CD55 positive) content of
the synovial tissue so that patients who responded to drug
treatment had a synovial lining layer structure approaching that of normal synovial tissue (17), as we have previously demonstrated (29). Although OPG content did increase with DMARD treatment, we have previously shown
that OPG is produced mainly by endothelial cells and
macrophages (16) rather than fibroblast lining cells, therefore changes in these cells in synovial tissue from patients
treated with DMARDs (both decreased) could not explain
the increase in OPG content of synovial tissue from these
patients.
Figure 1. Graphic representation of erosion and joint space narrowing scores (JSN; using the van der Heide modification of the
Sharp score [28]) over time for A, patients who responded well to
disease-modifying antirheumatic drug (DMARD) treatment and B,
patients with no response to DMARD treatment, based on the
Disease Activity Score in 28 joints. Solid circles ⫽ erosion score
hands; solid squares ⫽ erosion score feet; open squares ⫽ total
erosion score; open triangles ⫽ JSN hands; solid triangles ⫽ JSN
feet; open circles ⫽ total JSN.
Real-time PCR measurements on cDNA from synovial
tissue. Real-time quantitative PCR confirmed the results
demonstrated using immunohistochemical labeling of RA
synovial tissue. There was a decrease in RANKL detection
and an increase in OPG detection at the messenger RNA
(mRNA) level when the followup synovial biopsy was
compared with the initial synovial biopsy in RA patients
started on DMARD treatment (data not shown). Average
⌬⌬Ct for OPG was 0.57 for active RA patients and 1.11 for
inactive RA patients, while the average ⌬⌬Ct for RANKL
406 ⫾ 780
963 ⫾ 1,046
2,483 ⫾ 1,166
2,359 ⫾ 1,229
2,141 ⫾ 1,342
2,227 ⫾ 1,156
2,118 ⫾ 970
398 ⫾ 657
514 ⫾ 716
1,597 ⫾ 2,308
2,657 ⫾ 3,316
2,862 ⫾ 3,402
3,934 ⫾ 4,747
4,023 ⫾ 4,714
Baseline
0.25
0.5
1.0
1.5
2
3
6,432 ⫾ 8,220
5,935 ⫾ 8,096
4,980 ⫾ 7,262
3,246 ⫾ 6,669
3,109 ⫾ 5,919
2,118 ⫾ 4,827
1,097 ⫾ 2,808
IOD RANKL
41,354 ⫾ 34,198
37,911 ⫾ 25,541
27,383 ⫾ 16,892
18,210 ⫾ 15,464
14,392 ⫾ 13,041
15,495 ⫾ 13,241
14,082 ⫾ 14,832
Area CD68
IOD
plasma
cells
15,152 ⫾ 14,045 492 ⫾ 686 12,748 ⫾ 12,386
16,459 ⫾ 13,694 371 ⫾ 712 10,656 ⫾ 12,075
23,254 ⫾ 21,715 786 ⫾ 547 4,335 ⫾ 5,383
31,411 ⫾ 25,813 734 ⫾ 590 7,359 ⫾ 6,543
39,339 ⫾ 26,838 764 ⫾ 690 2,952 ⫾ 3,963
42,376 ⫾ 32,341 864 ⫾ 560 4,674 ⫾ 4,329
43,329 ⫾ 25,678 1,014 ⫾ 875 4,390 ⫾ 3,378
Area CD55
IOD B
cells
5,467 ⫾ 4,065
4,407 ⫾ 4,370
3,911 ⫾ 4,071
3,591 ⫾ 4,424
1,899 ⫾ 2,128
4,172 ⫾ 3,031
4,020 ⫾ 2,342
IOD T cells
IOD
granzyme
B cells
DAS28
9,801 ⫾ 8,546 23 ⫾ 15 5.5 ⫾ 0.6
8,744 ⫾ 6,789 30 ⫾ 26 4.2 ⫾ 1.9
8,170 ⫾ 5,669 278 ⫾ 234 2.5 ⫾ 2
5,911 ⫾ 5,390 192 ⫾ 205 1.8 ⫾ 1.5
4,793 ⫾ 4,975 158 ⫾ 207 1.5 ⫾ 1.2
4,419 ⫾ 3,593 43 ⫾ 21 1.5 ⫾ 1.2
4,082 ⫾ 4,779 63 ⫾ 70 1.5 ⫾ 1.3
IOD
memory
T cells
696 ⫾ 513
774 ⫾ 629
992 ⫾ 778
1,059 ⫾ 999
1,174 ⫾ 839
1,032 ⫾ 939
1,051 ⫾ 925
547 ⫾ 835
882 ⫾ 901
1,293 ⫾ 994
1,167 ⫾ 927
1,280 ⫾ 922
1,219 ⫾ 1,106
1,484 ⫾ 1,090
Baseline
0.25
0.5
1.0
1.5
2
3
7,372 ⫾ 7,260
7,115 ⫾ 7,508
5,145 ⫾ 5,708
5,123 ⫾ 6,239
4,912 ⫾ 6,374
4,925 ⫾ 6,362
5,667 ⫾ 3,664
IOD RANKL
* Values are the mean ⫾ SD. See Table 1 for definitions.
IOD 805
IOD 8051
Time of
synovial
biopsy
since
baseline,
years
22,665 ⫾ 19,702
21,555 ⫾ 19,754
22,826 ⫾ 19,871
26,909 ⫾ 19,460
18,678 ⫾ 16,548
17,967 ⫾ 20,156
18,018 ⫾ 15,291
Area CD68
23,172 ⫾ 22,329
23,648 ⫾ 24,015
22,478 ⫾ 27,290
22,646 ⫾ 26,192
22,950 ⫾ 26,095
26,060 ⫾ 26,670
27,863 ⫾ 29,001
Area CD55
434 ⫾ 453
427 ⫾ 423
135 ⫾ 209
115 ⫾ 216
157 ⫾ 226
160 ⫾ 225
200 ⫾ 248
IOD B
cells
8,904 ⫾ 942
8,722 ⫾ 759
9,091 ⫾ 8,085
5,989 ⫾ 5,410
5,887 ⫾ 5,410
6,019 ⫾ 7,393
7,362 ⫾ 7,126
IOD
plasma
cells
2,332 ⫾ 2,384
1,874 ⫾ 1,178
1,951 ⫾ 1,306
1,908 ⫾ 1,324
1,929 ⫾ 2,342
1,896 ⫾ 1,329
2,252 ⫾ 2,696
IOD T cells
8,498 ⫾ 9,004
8,432 ⫾ 9,973
6,721 ⫾ 6,779
5,318 ⫾ 6,961
5,329 ⫾ 5,961
5,333 ⫾ 6,543
6,309 ⫾ 5,718
IOD
memory
T cells
68 ⫾ 78
60 ⫾ 77
59 ⫾ 77
64 ⫾ 80
62 ⫾ 80
61 ⫾ 80
63 ⫾ 46
IOD
granzyme
B cells
6.3 ⫾ 0.7
6.0 ⫾ 1.2
5.5 ⫾ 1.0
4.7 ⫾ 1.1
4.8 ⫾ 1.1
4.7 ⫾ 1.3
4.5 ⫾ 1.5
DAS28
Table 2. Results of immunohistochemical labeling of synovial tissue from rheumatoid arthritis patients who demonstrated no clinical response to disease-modifying
antirheumatic drug treatment, based on a DAS28 <2.6*
* Values are the mean ⫾ SD. DAS28 ⫽ Disease Activity Score in 28 joints; IOD ⫽ integrated optical density, measured by image analysis; 8051 ⫽ monoclonal antibody that measures monomeric
osteoprotegerin (OPG) produced mainly by macrophage-like synovial lining cells; 805 ⫽ monoclonal antibody that measures dimeric OPG, mainly expressed by endothelial cells; CD68 ⫽ monoclonal
antibody that detects macrophage lineage cells; CD55 ⫽ monoclonal antibody that detects decay activator factor, a marker for synovial lining fibroblasts.
IOD 805
IOD 8051
Time of
synovial
biopsy
since
baseline,
years
Table 1. Results of immunohistochemical labeling of synovial tissue from rheumatoid arthritis patients with a good clinical response to disease-modifying antirheumatic
drug treatment based on a DAS28 <2.6*
RANKL and OPG in RA Synovial Tissue
915
916
Haynes et al
Figure 2. Immunohistochemical labeling of sequential synovial biopsy samples from the same knee joint in a patient with rheumatoid
arthritis obtained at A, baseline, B, 3 months, C, 6 months, and D, 12 months after starting methotrexate. Biopsy samples are labeled with
monoclonal antibody 805, detecting osteoprotegerin in blood vessels, with aminoethylcarbazole as the chromogen (red color). (Original
magnification ⫻ 400.)
was 1.31 for active RA patients and 0.18 for inactive RA
patients. This confirmed the results for the immunohistochemical labeling studies at the mRNA level.
Correlation of immunohistochemical labeling for
RANKL and OPG with changes in clinical and radiologic
parameters. Although there was an increase in synovial
tissue expression of OPG with DMARD treatment, the correlation with change in DAS28 failed to achieve statistical
significance (r ⫽ 0.402, P ⫽ 0.052). The decrease in synovial tissue expression of RANKL (measured by either image analysis or semiquantitative score) did correlate with
the reduction in DAS28 with DMARD treatment (r ⫽
0.448, P ⫽ 0.037). There was also a significant correlation
between changes in DAS28 and changes in joint space
narrowing score (r ⫽ 0.454, P ⫽ 0.023), with the least
change in joint space narrowing score seen in patients
with the greatest reduction in DAS28. Similarly, there was
a significant correlation between changes in RANKL expression in synovial tissue (measured with either image
analysis or semiquantitative score) and changes in erosion
score (r ⫽ 0.538, P ⫽ 0.007), with the smallest changes in
erosion score seen in patients with the greatest reduction
in RANKL expression in synovial tissue following
DMARD treatment. There was no correlation of changes in
either RANKL or OPG expression in the synovial tissue
with changes in joint space narrowing with DMARD treatment. We did not demonstrate any effect of disease duration on changes in OPG or RANKL seen with DMARD
treatment. We attempted to calculate RANKL:total OPG,
RANKL:endothelial OPG, and RANKL:synovial lining
OPG ratios, but the data were skewed in distribution and
neither logarithmic nor inverse transformation of the data
led to a meaningful analysis.
DISCUSSION
Osteoclasts are responsible for the resorption of bone during normal bone metabolism and the destruction of bone
seen in a variety of pathologies such as RA. It is now clear
RANKL and OPG in RA Synovial Tissue
917
Figure 3. Immunohistochemical labeling of sequential synovial biopsy samples from the same knee joint in a patient with rheumatoid
arthritis obtained at A, baseline, B, 3 months, C, 6 months, and D, 12 months after starting methotrexate. Biopsy samples are labeled with
monoclonal antibody 8051, detecting osteoprotegerin in the synovial lining, with aminoethylcarbazole as the chromogen (red color).
(Original magnification ⫻ 400.)
that M-CSF and RANKL are essential factors required for
the development of osteoclasts. Osteoclasts form from
cells isolated from the RA joint, with large numbers rapidly forming from cells isolated from the pannus region
(8). The cartilage–pannus junction in RA contains many
types of cells that produce inflammatory cytokines reported to stimulate osteoclast differentiation and bone resorption, including IL-1␣ and IL-1␤, IL-6, IL-11, and TNF␣
(9). The end result of the production of inflammatory cytokines, such as IL-1␤ and TNF␣, in the inflamed joint is
likely to be the up-regulation of RANKL (produced by T
cells, fibroblasts, and osteoblasts) (8,13) and RANK (expressed by preosteoclasts, T cells, and dendritic cells) (13).
OPG is an alternative, high-affinity decoy receptor for
RANKL that blocks the interaction between RANKL and
RANK and significantly inhibits osteoclastogenesis
(14,16). Similar to RANK and RANKL, OPG production is
stimulated in vitro by proinflammatory cytokines, such as
IL-1␤ and TNF␣. RANK, RANKL, and OPG are expressed
in synovial tissue from the RA joint (5,6,8,15,16).
We have previously demonstrated the expression of
OPG in synovial tissue (both lining and endothelial expression) from patients with both inflammatory arthritis
and osteoarthritis, as well as normal synovial tissue (16).
The notable exception to this was in RA synovial tissue:
little or no OPG was expressed in the synovial tissue from
patients with active RA, whereas OPG was expressed both
in the synovial lining and on endothelial cells in the
synovial tissue from patients with inactive RA. In contrast,
we have also demonstrated the expression of RANKL
mainly in the synovial tissue of patients with active RA
(15). This leads to a local synovial environment in the
active RA joint where osteoclast formation would be
likely, whereas in the inactive RA joint osteoclast formation would be suppressed. The current study was designed
to test this hypothesis by examining the expression of
RANKL and OPG at the synovial tissue level in patients
with active RA started on treatment with DMARDs and by
attempting to correlate changes in synovial expression of
RANKL and OPG with radiologic outcomes for this RA
918
Haynes et al
Figure 4. Immunohistochemical labeling of sequential synovial biopsy samples from the same knee joint in a patient with rheumatoid
arthritis obtained at A, baseline, B, 3 months, C, 6 months, and D, 12 months after starting methotrexate. Biopsy samples are labeled with
a monoclonal antibody detecting RANKL, with aminoethylcarbazole as the chromogen (red color). (Original magnification ⫻ 400; insets,
original magnification ⫻ 800.)
patient cohort. Because it was not predictable if and when
an individual patient would respond to DMARD treatment
and when any change in synovial membrane expression of
RANKL or OPG would occur, synovial biopsies were performed up to 36 months after initiation of treatment, if the
patient remained in the study. Our study is the first to
demonstrate that successful DMARD treatment of patients
with RA can result in the reduction of RANKL expression
and an increase in OPG expression at the synovial tissue
level, with a resultant reduction in the RANKL:OPG ratio,
which would reduce the likelihood of osteoclast formation
and, theoretically, joint erosion and joint space narrowing.
We have confirmed these results at the mRNA level using
a subset of RA patients included in this study. Our study is
also the first to demonstrate that there is a correlation
between changes in DAS28 and both synovial expression
of RANKL and OPG as well as joint erosion measured
radiologically, as a result of successful DMARD treatment.
Although we had a high rate of response to conventional
DMARD treatment (52% with low disease activity based
on a DAS28), it should be noted that 72% of the patients
included in this study had a disease duration ⬍1 year at
study entry. The early-disease RA population has previously been shown to have a higher and similar response
rate to conventional DMARDs and biologic treatments
(31,32). Similarly, our patient population had a relatively
high erosion rate at study entry, but previous studies have
shown that this is not unusual, with most erosions being
detectable within the first 2 years of disease (33,34). Our
RA patient population is also unusual in that there were
more men than women. Although sex may have some
effect on clinical and radiologic outcomes, it is small compared with the effect that the presence of rheumatoid
factor (or anti– cyclic citullinated peptide antibodies) and
erosions on radiograph at disease onset has on these outcomes. Despite the above caveats, we believe that our
results are generalizable to the global rheumatology population.
A recent study by Catrina et al (35) also demonstrated
that OPG expression was low in RA synovial tissue while
RANKL and OPG in RA Synovial Tissue
RANKL expression was high in patients with active disease, and that treatment with TNF blockers (either etanercept or infliximab) altered the RANKL:OPG ratio in favor
of a reduction in osteoclast formation. However, unlike
our previous findings, these authors failed to find any
correlation between changes in disease activity and
changes in synovial tissue expression of RANKL and OPG,
and suggested that the changes they observed were a direct
result of TNF blockade. The patient group included in the
study by Catrina et al was similar to that used in our study,
but Catrina et al did not indicate the disease duration of
their patient group, so it is not possible to establish
whether the RA patients they studied had early RA, as did
the majority of the patients included in our study. In
addition, there was considerable coprescription of both
corticosteroids and DMARDs (mainly methotrexate) with
infliximab and etanercept in their study, so it is unclear
whether the effects on OPG and RANKL expression in RA
synovial tissue were solely due to TNF inhibitors. Finally,
Catrina et al only followed patients for 8 weeks and the
clinical responses seen in these patients (ACR20 responses
and reduction in DAS28 scores to mean levels of 3.8 [etanercept] and 4.2 [infliximab]) were inferior to those seen
in the patients included in our study. A further study by
the same research group (36) demonstrated that intraarticular corticosteroids could significantly reduce the synovial
content of CD3-positive T lymphocytes and the expression
of RANKL by these cells without affecting synovial expression of OPG, potentially reducing the risk of erosions in
the RA joint, although the researchers did not measure the
effect on radiologic outcomes. While these authors did not
demonstrate any effect of intraarticular corticosteroids on
synovial tissue OPG expression, the antibody they used
detects only OPG on endothelial cells and not on macrophages, as we have previously demonstrated (16).
The results of our study suggest that a deficiency in OPG
expression may have a role in the pathogenesis of bone
erosions, which characterize RA, and suggest that OPG
may well have a therapeutic role in the future management
of RA. Standard DMARD treatment of patients with RA
can down-regulate RANKL expression in RA synovial tissue with significant implications for progression of erosive
damage within joints over time. The development of treatments for RA that modulate both the inflammatory milieu
of the synovial tissue and the mediators of osteoclast formation is likely to result in significant improvement in the
clinical, functional, and radiologic outcomes for patients
with RA.
AUTHOR CONTRIBUTIONS
Dr. Smith had full access to all of the data in the study and takes
responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Haynes, Crotti, Smith.
Acquisition of data. Haynes, Crotti, Weedon, Slavotinek, Au,
Ahern, Smith.
Analysis and interpretation of data. Haynes, Crotti, Coleman,
Ahern, Smith.
Manuscript preparation. Haynes, Crotti, Roberts-Thomson,
Ahern, Smith.
Statistical analysis. Ahern.
919
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expressions, treatment, patients, modifying, disease, tissue, modulation, osteoprotegerin, antirheumatic, outcomes, correlation, radiological, drug, response, arthritis, synovial, rank, rheumatoid
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