Intraarticular corticosteroids decrease synovial RANKL expression in inflammatory arthritis.
ABSTRACT Intraarticular corticosteroids are frequently used as successful adjuvant therapy for inflammatory arthritides, but little is known about their effects on molecules that regulate bone biology. We undertook this study to investigate the effect of intraarticular corticosteroids on the synovial expression of RANKL and osteoprotegerin (OPG).
We evaluated RANKL, OPG, and surface marker expression by immunohistochemical methods in synovial knee biopsy samples obtained from 13 patients with inflammatory arthritis before and 2 weeks following intraarticular injection of triamcinolone hexacetonide. We further investigated the effect of dexamethasone (DEX) on RANKL expression by lymphocytes from rheumatoid arthritis synovial fluids (RA SF), using flow cytometric analysis. Finally, we evaluated the in vitro effect of DEX on RANKL and OPG expression in osteoblast-like cells, by Western blotting.
Intraarticular corticosteroids induced a decrease in the number of synovial T cells without influencing the number of macrophages, evaluated as both CD68+ and CD163+ cells. This change was paralleled by a decrease of synovial RANKL expression with a concomitant reduction of the RANKL:OPG ratio. DEX down-regulated RANKL expression on lymphocytes derived from RA SF. Moreover, in vitro pretreatment of osteoblast-like cells with tumor necrosis factor favored an antiresorptive effect of DEX treatment through a similar down-regulation of RANKL expression.
The decrease in inflammation attributed to intraarticular corticosteroids is accompanied by down-modulation of bone destruction markers. These findings offer a rationale for the beneficial effect of corticosteroids on joint erosion in arthritis.
- SourceAvailable from: Krishnadas Madhu[Show abstract] [Hide abstract]
ABSTRACT: Aim Rheumatoid arthritis is a systemic inflammatory disease, which mainly affects joints but can also affect other organs. Ocimum gratissimum L. (Lamiaceae) is a herb, which is reported to have anti-inflammatory activity. So the present investigation was designed to evaluate antiarthritic potential of O. gratissimum in Collagen induced arthritis (CIA) in laboratory animals. Materials and methods Fresh leaves were extracted using 95% ethanol by maceration process. The extracts were tested against collagen induced female Sprague-Dawley rats. Arthritis assessment was carried out on the basis of parameters including arthritic score, body weight and paw volume. On day 41, animals were evaluated for biochemical parameter, radiological parameter and histological parameters. Results Treatment with 500 mg/kg of EEOG significantly reduced arthritic score, paw volume and almost restored body weight of arthritic animals compared to control animals. Meanwhile, the treatment significantly attenuated biochemical, radiological and histopathological alteration induced by CIA. Conclusion EEOG can able to nullify most of the ill effects produced by CIA in rats. However, further studies are needed to carry out identification, isolation of active fractions of EEOG to unravel the mechanism of drug.Biomedicine & Aging Pathology. 07/2014;
- [Show abstract] [Hide abstract]
ABSTRACT: Increased bone resorption and enhanced risk of osteoporotic fractures are often reported in patients with diseases having immune system involvement, mainly inflammatory rheumatic diseases, for which glucocorticoids are more often prescribed because of their powerful antinflammatory effects. Among secondary osteoporosis, glucocorticoid-induced osteoporosis is the most common and severe form, and up to now prolonged glucocorticoid therapy has been considered the most important osteoporotic risk factor in rheumatic patients.However, it is now clear that the pathogenesis of osteoporosis in inflammatory rheumatic diseases is mediated by several factors. In particular, new discoveries within osteoimmunology concerning the complex relationship between bone and immunity, suggest that inflammation itself, through proinflammatory and osteoclastogenic cytokine overexpression, promotes bone resorption leading to increased skeletal fragility. Therefore, by controlling systemic inflammation, it is also possible to reduce the loss of bone mass which accompanies rheumatic diseases.From this point of view, we critically revisit the effects of glucocorticoids on bone in rheumatic diseases, in which they should be seen not just as an enemy of the bone health but eventually as a potential therapeutic tool capable of reducing the risk of osteoporosis by controlling disease activity and inflammation.Current Medicinal Chemistry 11/2014; · 3.72 Impact Factor
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ABSTRACT: The inflammatory joint diseases of juvenile inflammatory arthritis (JIA), rheumatoid arthritis (RA) and osteoarthritis (OA): and also mild to moderate joint injury, all require a multidisciplinary approach to management. Intra-articular injections of corticosteroids have been shown to be a very beneficial adjunctive treatment in the management of the above disorders. It is, therefore, important that clinicians have a good understanding of the clinical actions of intra-articular injections. This article explores the pharmacokinetics, pharmacodynamics, and clinical pharmacology of triamcinolone acetonide (TA) and triamcinolone hexacetonide (TH) in JIA, RA, and OA. Literature search of TA and TH articles was conducted using key word searches in the PubMed and Google Scholar databases and through references within found articles. TA and TH intra-articular injections have been shown to provide good clinical benefit for up to 6 months and even longer. TH has been shown to decrease in the expression of citrullinated proteins, the monoclonal antibody F95, and peptidylarginine deiminase 4 in RA synovium. TA and TH intra-articular injections have a low side effect profile which is similar to other corticosteroid. They have minimal to no mineralocorticoid adverse effects and facial flushing 2-3 days post injections is the most common side effect recorded, and in almost all cases is no worse than nuisance. TA and TH are useful adjunct therapies in the management of JIA, RA, OA, and mild to moderate joint injury.Inflammopharmacology 05/2014; 22(4).
ARTHRITIS & RHEUMATISM
Vol. 54, No. 5, May 2006, pp 1463–1472
© 2006, American College of Rheumatology
Intraarticular Corticosteroids Decrease Synovial RANKL
Expression in Inflammatory Arthritis
Dimitrios Makrygiannakis,1Erik af Klint,1Sergiu-Bogdan Catrina,2Ileana Ruxandra Botusan,2
Elin Klareskog,1Lars Klareskog,1Ann-Kristin Ulfgren,1and Anca Irinel Catrina1
Objective. Intraarticular corticosteroids are fre-
quently used as successful adjuvant therapy for inflam-
matory arthritides, but little is known about their effects
on molecules that regulate bone biology. We undertook
this study to investigate the effect of intraarticular
corticosteroids on the synovial expression of RANKL
and osteoprotegerin (OPG).
Methods. We evaluated RANKL, OPG, and sur-
face marker expression by immunohistochemical meth-
ods in synovial knee biopsy samples obtained from 13
patients with inflammatory arthritis before and 2 weeks
following intraarticular injection of triamcinolone
hexacetonide. We further investigated the effect of dexa-
methasone (DEX) on RANKL expression by lympho-
cytes from rheumatoid arthritis synovial fluids (RA SF),
using flow cytometric analysis. Finally, we evaluated the
in vitro effect of DEX on RANKL and OPG expression
in osteoblast-like cells, by Western blotting.
Results. Intraarticular corticosteroids induced a
decrease in the number of synovial T cells without
influencing the number of macrophages, evaluated as
both CD68? and CD163? cells. This change was par-
alleled by a decrease of synovial RANKL expression
with a concomitant reduction of the RANKL:OPG ratio.
DEX down-regulated RANKL expression on lympho-
cytes derived from RA SF. Moreover, in vitro pretreat-
ment of osteoblast-like cells with tumor necrosis factor
favored an antiresorptive effect of DEX treatment
through a similar down-regulation of RANKL expres-
Conclusion. The decrease in inflammation attrib-
uted to intraarticular corticosteroids is accompanied by
down-modulation of bone destruction markers. These
findings offer a rationale for the beneficial effect of
corticosteroids on joint erosion in arthritis.
Inflammatory arthritides such as rheumatoid ar-
thritis (RA) might result in local bone destruction
through osteoclast recruitment at the inflammation site
(1). This process is regulated through the RANKL,
RANK, and osteoprotegerin (OPG) system (2).
RANKL, a member of the tumor necrosis factor
(TNF) ligand superfamily, is mainly secreted by osteo-
blasts (3) and activated T cells (4) following stimulation
with proinflammatory cytokines such as TNF and
interleukin-1 (5). RANKL interacts with its receptor
RANK on the surface of premature osteoclasts, inducing
osteoclast formation, activation, and survival (6). Be-
sides its role in osteoclastogenesis, RANKL acts as an
immune mediator, promoting activation of monocytes
(7) and interactions between dendritic cells and T cells
(8). OPG, a member of the TNF receptor superfamily, is
a soluble decoy receptor with the ability to block the
interaction between RANKL and RANK (6). A high
RANKL:OPG ratio is associated with increased bone
resorption (9) and with active synovitis (10).
Intraarticular corticosteroids are potent antiin-
flammatory compounds that are successfully used by
clinicians as an adjuvant therapy for inflammatory ar-
thritis. One of the first randomized controlled studies
regarding corticosteroid use in RA demonstrated a
Supported by the Swedish Medical Research Council, the
Swedish Rheumatism Association, the AMF Insurance Company, and
King Gustaf V’s 80-Year Foundation. Dr. Botusan’s work was sup-
ported by the Marie Curie fellowship within the Eurogendis program.
1Dimitrios Makrygiannakis, MD, Erik af Klint, MD, Elin
Klareskog, Lars Klareskog, MD, PhD, Ann-Kristin Ulfgren, PhD,
Anca Irinel Catrina, MD, PhD: Karolinska University Hospital, Solna,
Karolinska Institutet, Stockholm, Sweden;
MD, PhD, Ileana Ruxandra Botusan, MD: Karolinska University
Hospital, Solna, Karolinska Institutet, Stockholm, Sweden, and Carol
Davila University of Medicine, Bucharest, Romania.
Drs. Makrygiannakis and af Klint contributed equally to this
Address correspondence and reprint requests to Anca Irinel
Catrina, MD, PhD, Rheumatology Department, Karolinska University
Hospital, Solna, Karolinska Institutet, CMM, L8-04, S-17176 Stock-
holm, Sweden. E-mail: email@example.com.
Submitted for publication June 15, 2005; accepted in revised
form January 12, 2006.
positive effect not only regarding clinical symptoms, but
also regarding joint erosion (11,12). Subsequent reports
have been somewhat contradictory (13), opening a still
valid debate regarding the real effect of corticosteroids
on joint destruction, even though relatively recent data
suggest once again a positive effect of corticosteroid
treatment in preventing bone erosion in RA (14). Nota-
bly, there are no reports from randomized clinical trials
on the effects of intraarticular corticosteroids on joint
erosion. Thus, even though corticosteroids are accepted
by clinicians as a powerful adjuvant therapy, there are
still concerns regarding the balance between their two
potentially divergent effects (bone protective versus
The mechanisms underlying the effects of intra-
articular corticosteroids are still not fully understood. In
order to contribute to such knowledge, we investigated
the effect of intraarticular corticosteroids on synovial
expression of RANKL and OPG. We characterized
RANKL and OPG expression by CD3? and CD163?
cells. We further evaluated the effect of corticosteroids
on RANKL expression by lymphocytes derived from RA
synovial fluid (SF). Finally, we investigated the effect
of corticosteroids on RANKL and OPG expression in
cultured osteoblast-like cells.
PATIENTS AND METHODS
Patients. Thirteen patients (11 women and 2 men,
median age 55 years, range 28–83 years) with active knee
arthritis (mean duration of current knee arthritis episode 3
months, mean disease duration 43 months) with different
pathogenesis (6 patients with RA, 2 patients with spondyl-
arthropathies, and 5 patients with unclassified oligoarthritis)
were recruited for this study. All patients received an intra-
articular injection of 40 mg of triamcinolone hexacetonide.
Synovial biopsy samples from areas close to cartilage were
obtained prior to and 2 weeks after the injection. All other
associated treatments (including disease-modifying drugs, bio-
logic agents, nonsteroidal antiinflammatory drugs, and oral
corticosteroids) were maintained at constant levels for at least
2 weeks before and throughout the whole study period.
During arthroscopy the joint cavity was carefully in-
spected, with representative pictures being kept as a record. A
semiquantitative scale was used by 2 independent observers to
evaluate synovial vascularity (0 ? scarce presence of vessels;
1 ? mild vascularity; 2 ? moderate vascularity; 3 ? prominent
vascularity) and hypertrophy (0 ? no villi; 1 ? mild hypertro-
phy; 2 ? moderate hypertrophy; 3 ? prominent hypertrophy).
Additionally, we investigated synovial biopsy samples obtained
during open surgery from 3 RA patients undergoing knee
The Ethics Committee at the Karolinska Hospital
approved all experiments on human cells and tissues. Informed
consent was obtained from all study subjects.
Tissue preparation and immunohistochemical analy-
sis. Serial cryostat sections (7 ?m) were fixed for 20 minutes
with 2% (volume/volume) formaldehyde or for 10 minutes with
100% acetone and stored at ?70°C. We evaluated synovial
expression of OPG and RANKL by immunohistochemistry,
using as primary antibodies mouse IgG1 anti-human OPG
antibody (MAB805) at a concentration of 2.5 ?g/ml and mouse
IgG2b anti-human RANKL antibody (MAB626) at a concen-
tration of 5.0 ?g/ml (both from R&D Systems, Minneapolis,
MN). We characterized the synovial cell phenotype using as
primary antibodies mouse IgG1 anti-human CD3 (SK7; BD
Biosciences, San Jose, CA), mouse IgG1 anti-human CD68
(KP1; DakoCytomation, Glostrup, Denmark), and mouse
IgG1 anti-human CD163 (Ber-MAC3; DakoCytomation), as
previously described (15). Matched IgG isotype controls were
included for all markers.
Immunofluorescence staining. Following avidin–biotin
blocking (SP-2001; Vector, Burlingame, CA), 2% formaldehyde–
fixed sections were incubated with the primary anti-RANKL or
anti-OPG antibodies overnight. Secondary biotinylated goat
anti-mouse antibody (BA2001; Caltag, Burlingame, CA) or
horse anti-mouse antibody (M32515; Vector) was added, fol-
lowed by the addition of streptavidin–Alexa Fluor 488 conju-
gate (S-11223; Molecular Probes, Eugene, OR). Sections were
further incubated with phycoerythrin (PE)–conjugated mouse
IgG1 monoclonal anti-human CD163 antibody (215927; R&D
Systems) or PE-conjugated mouse IgG1 monoclonal anti-
human CD3 antibody (345765; BD Biosciences) and mounted
with Mowiol 4-88 mounting medium (475904; Calbiochem, San
Diego, CA). All antibodies and fluorophores were diluted in
phosphate buffered saline (PBS)/0.1% saponin/0.1% bovine
serum albumin (BSA). Matched isotype negative controls were
Microscopic analysis. Stained synovial biopsy sections
were evaluated semiquantitatively, using a 4-point scale, by 2
independent observers (AIC and DM) who were unaware of
the patient’s identity and biopsy sequence. The final scores
represent the means of the 2 observations. For quantification,
synovial expression of each marker was evaluated, using
computer-assisted image analysis, by a single observer (DM)
who was unaware of the patient identity of each section (mean
of 52 microscopic fields, 250? magnification), and the results
were expressed as the percentage of total tissue area that
stained positive. Cell counting of RANKL? cells and CD3?
cells was performed on serial sections, and the results were
expressed as the percentage of CD3? cells that were
SF preparation and flow cytometric analysis. SF mono-
nuclear cells (SFMCs) from 3 RA patients were cultured in
triplicate in RPMI supplemented with 2 mM L-glutamine, 50
IU/ml penicillin and 50 ?g/ml streptomycin, and 20% heat-
inactivated fetal calf serum (FCS) (all from Gibco, Paisley,
UK) and incubated at 37°C in a humidified atmosphere con-
taining 5% CO2.Where indicated, dexamethasone (DEX,
861871; Sigma, St. Louis, MO) was added to a final concen-
tration of 1, 10, 100, or 1,000 nM and incubated for 24 hours.
To determine whether DEX decreases RANKL expression,
cells were double-stained with PE-conjugated mouse IgG1
anti-CD3 (BD Biosciences) and mouse IgG2b anti-RANKL
(MAB626) followed by fluorescein isothiocyanate–conjugated
rabbit anti-mouse secondary antibody (BD Biosciences). Scat-
1464 MAKRYGIANNAKIS ET AL
ter properties were used to identify the lymphocyte population.
Results were expressed as the percentage of the total number
of gated lymphocytes that were RANKL?. Isotype controls
using mouse IgG2b (DakoCytomation) were included in each
Cell culture and Western blotting analysis. Human
osteoblast-like cells (SaOS-2) (ACC 243; DSMZ, Braun-
schweig, Germany) were grown in a humidified atmosphere
with 5% CO2at 37°C in McCoy’s 5A medium supplemented
with 2 mM L-glutamine, 100 IU/ml penicillin and streptomycin,
and 5% heat-inactivated FCS (all culture reagents were pur-
chased from Gibco). Cells were maintained for 24 hours
without FCS and incubated for 24 hours with or without 1 nM
recombinant human TNF (210-TA; R&D Systems). Follow-
ing incubation, cells were washed with PBS and incubated for
24 hours with or without DEX. At the end of the incubation
period, a whole-cell protein extract was prepared as pre-
viously described (16). Following sodium dodecyl sulfate–
polyacrylamide gel electrophoresis, 50 ?g of total cellular
proteins (measured using the Bradford assay and BSA stan-
dards) were Western blotted on a nitrocellulose filter and
blocked overnight with 5% nonfat milk in PBS. The membrane
Figure 1. Arthroscopic images illustrating the joints of a patient with rheumatoid arthritis before (A) and after (B) intraarticular corticosteroid
injection, of an oligoarthritis patient before (C) and after (D) intraarticular corticosteroid injection, and of a spondylarthropathy patient before (E)
and after (F) intraarticular corticosteroid injection.
EFFECT OF INTRAARTICULAR CORTICOSTEROIDS ON RANKL EXPRESSION1465
was incubated for 2 hours either with anti-human RANKL or
with OPG primary antibody at a concentration of 2 ?g/ml in
PBS containing 1% nonfat milk. After washing, the membrane
was incubated for 1.5 hours with the secondary peroxidase-
linked anti-mouse IgG (NXA93; Amersham Biosciences,
Buckinghamshire, UK) diluted in PBS containing 1% nonfat
After washing, the complexes were visualized using
Figure 2. CD3? synovial T cells express RANKL but not osteoprotegerin (OPG), while CD163? synovial macrophages express neither RANKL
nor OPG in rheumatoid arthritis synovium. A, Photomicrographs illustrating fluorescent staining of CD3? cells (red, phycoerythrin), OPG? cells
(green, Alexa Fluor 488), and superimposed stainings. B, Photomicrographs illustrating fluorescent staining of CD3? cells (red, phycoerythrin),
RANKL? cells (green, Alexa Fluor 488), and superimposed stainings. C, Photomicrographs illustrating fluorescent staining of CD163? cells (red,
phycoerythrin), OPG? cells (green, Alexa Fluor 488), and superimposed stainings. D, Photomicrographs illustrating fluorescent staining of CD163?
cells (red, phycoerythrin), RANKL? cells (green, Alexa Fluor 488), and superimposed stainings. (Original magnification ? 250 in A and B; ? 125
in C and D.)
1466 MAKRYGIANNAKIS ET AL
enhanced chemiluminescence (RPN2209; Amersham Bio-
sciences). X-ray films were analyzed using a computerized
image analysis system (Kodak 1D 3.5; Eastman Kodak, Roch-
ester, NY). Results were expressed as arbitrary units repre-
senting the ratio of the net intensity of the RANKL band to
the net intensity of the OPG band corrected for ?-actin
Statistical analysis. Statistical analysis was performed
using the Wilcoxon test followed by Bonferroni correction for
multiple comparisons of paired samples. Differences between
groups were analyzed using the Mann-Whitney U test. In vitro
results were analyzed by one-way analysis of variance with
Tukey post hoc analysis test. P values less than 0.05 were
Clinical response and arthroscopic evaluation.
All patients included in the study were clinical respond-
ers as evaluated by physician’s assessment. Macroscopic
evaluation of the investigated joints demonstrated a
significant decrease in synovial inflammation 2 weeks
after intraarticular corticosteroid injection (Figure 1).
The mean ? SEM score for synovial vascularity de-
creased from 2.4 ? 0.2 to 0.8 ? 0.1, and that for synovial
hypertrophy decreased from 1.4 ? 0.3 to 0.8 ? 0.2. We
did not observe significant differences between the
different diseases studied.
Histologic evaluation of synovial expression of
OPG and RANKL. We first characterized the synovial
distribution pattern of both RANKL and OPG in syno-
vial samples obtained during open surgery in 3 RA
patients and by arthroscopy in 4 arthritis patients (1 with
oligoarthritis, 2 with RA, and 1 with spondylarthro-
pathy). Synovial OPG was mainly detected in endothe-
lial cells, with no expression either in CD3? T cells
(Figure 2A) or in CD163? macrophages (Figure 2C) as
evaluated by double-staining. In contrast, RANKL was
Figure 3. Reduction of synovial cellularity by intraarticular corticosteroids. Frozen sections of synovial biopsy tissues from 1 patient treated with
intraarticular corticosteroids show diaminobenzidine (brown) immunoperoxidase staining (hematoxylin counterstained) for surface markers CD3,
CD68, and CD163 before and after treatment (original magnification ? 250). Graphs show results from image analysis of biopsy tissues for CD3,
CD68, and CD163 before and after intraarticular corticosteroid injection. Values are the mean ? SEM. ? ? P ? 0.05 versus before treatment.
EFFECT OF INTRAARTICULAR CORTICOSTEROIDS ON RANKL EXPRESSION 1467
mainly present on small lymphocyte-like cells in T cell
areas. Double-labeling with either CD3 and RANKL
(Figure 2B) or CD163 and RANKL (Figure 2D) demon-
strated that T cells, but not macrophages, expressed
Synovial histologic changes following intraartic-
ular corticosteroid injection. We then wanted to deter-
mine whether the clinical response observed in all
treated patients was paralleled by a change in synovial
histology. We were able to demonstrate that intraartic-
ular corticosteroids induced a significant decrease in the
number of synovial T cells, as evaluated by CD3 staining,
without influencing the number of macrophages, evalu-
ated as both CD68? and CD163? cells (Figure 3).
The decrease in the T cell number was accompa-
nied by a significant reduction in synovial RANKL
expression (Figure 4). This was not merely a secondary
measurement of the lower number of T cells following
Figure 4. Reduction of synovial RANKL expression and synovial RANKL:osteoprotegerin (OPG) ratio by intraarticular corticosteroids. Frozen
sections of synovial biopsy tissues from 1 patient treated with intraarticular corticosteroids show diaminobenzidine (brown) immunoperoxidase
staining (hematoxylin counterstained) for RANKL and OPG before and after treatment (original magnification ? 250). Graphs show results from
image analysis of biopsy tissues for RANKL and OPG, as well as the synovial RANKL:OPG ratio, before and after intraarticular corticosteroid
injection. Values are the mean ? SEM. ? ? P ? 0.05 versus before treatment.
1468MAKRYGIANNAKIS ET AL
treatment, since the number of CD3? T cells expressing
RANKL also decreased significantly (from a mean ?
SEM of 32.27 ? 7.51% to a mean ? SEM of 8.71 ?
3.51%) (P ? 0.05). We did not observe any changes in
synovial OPG expression following corticosteroid injec-
tion (Figure 4). The decrease in RANKL expression
resulted in a significant reduction of the synovial
RANKL:OPG ratio, from a mean ? SEM of 0.18 ? 0.05
to a mean ? SEM of 0.03 ? 0.01 (P ? 0.05) (Figure 4).
Corticosteroids decrease RANKL expression in
SF-derived lymphocytes. To further evaluate the effect of
corticosteroids, we investigated RANKL expression in
Figure 5. Dexamethasone (DXM) decreases RANKL expression in synovial fluid–derived lymphocytes from patients with rheumatoid arthritis. A,
Percentage of lymphocytes that were RANKL?. Values are the mean and SEM. ? ? P ? 0.05 versus no dexamethasone treatment. B, Histogram
demonstrating a decrease in RANKL staining in synovial fluid mononuclear cell–derived lymphocytes following treatment with dexamethasone.
Black line represents control untreated sample; gray line represents dexamethasone-treated sample.
Figure 6. Dexamethasone (DXM) attenuates tumor necrosis factor (TNF)–induced changes in RANKL/osteoprotegerin (OPG) expression in
osteoblast-like cells, as evaluated by Western blotting. A, Dexamethasone (100 nM) induces RANKL and decreases OPG expression in osteoblast-like
cells following 24 hours of incubation. B, Dexamethasone (100 nM) decreases TNF-induced RANKL expression in TNF-primed osteoblast-like cells
following 24 hours of incubation, without influencing OPG expression. C, RANKL:OPG ratio in osteoblast-like cells as evaluated by Western
blotting and subsequent computer image analysis. Values are the mean and SEM. ? ? P ? 0.05. D, Dexamethasone induces a dose-dependent
decrease of RANKL expression in TNF-primed osteoblast-like cells, as evaluated by Western blotting. C ? control.
EFFECT OF INTRAARTICULAR CORTICOSTEROIDS ON RANKL EXPRESSION1469
SFMCs in relation to DEX treatment. We demonstrated
that DEX could significantly decrease the surface expres-
sion of RANKL in the lymphocyte population of RA SF at
dosages as low as 1 nM (Figures 5A and B).
Corticosteroids attenuate TNF-dependent induc-
tion of RANKL in osteoblasts. Since bone is the target
organ of the RANKL/OPG system, we sought to inves-
tigate the possible relevance of our findings in an in vitro
model using osteoblast-like cells. Treatment of osteo-
blasts with DEX promoted osteoclastogenesis by inhib-
iting OPG and increasing RANKL expression in
osteoblast-like cells (Figure 6A). However, following
TNF priming, a condition that mimics the proinflamma-
tory milieu of the rheumatoid joint, DEX decreased
RANKL expression in a dose-dependent manner (Fig-
ures 6B and D), resulting in a significant down-
regulation of the RANKL:OPG ratio (Figure 6C).
Despite the fact that intraarticular corticosteroids
represent a longstanding adjuvant treatment of inflamma-
tory arthritis, the molecular mechanisms of their action are
not yet fully understood. Here we demonstrate that one
candidate mechanism to explain their clinical benefits,
particularly concerning the potential effects on joint de-
struction, is modulation of the synovial RANKL/OPG
system. We propose that the decrease of the synovial
RANKL:OPG ratio following intraarticular cortico-
steroid injection represents a plausible mechanism for the
bone-protective effect described in reports of previous
studies using systemic corticosteroid therapy (17,18).
Since their first use as a treatment for RA more
than 50 years ago, corticosteroids have been the subject
of clinical debate based on their potential deleterious
effect on bone density and bone function, as well as their
potential positive effects on joint destruction. Concern-
ing the deleterious effects, secondary osteoporosis rep-
resents an important limitation of long-term systemic
treatment with high doses of corticosteroids (19). This
observation raised concerns regarding their use in RA, a
disease characterized by bone destruction. One of the
first placebo-controlled studies recorded a positive ef-
fect on radiographic evolution 3 years after cortico-
steroid treatment (11,12), while a report of another early
study claimed no such effect (13). Subsequent placebo-
controlled studies in early RA, with or without associ-
ated treatment, demonstrated that oral corticosteroids
retard the progression and occurrence of radiographic
damage in RA patients (14,20–23). Even though few
data are available regarding intraarticular corticosteroids
(17,24), it has been proposed that local corticosteroids
might contribute to local disease control (i.e., to the
slowing of damage progression in injected joints) (25).
In our group of patients, treatment with cortico-
steroids reduced signs and symptoms of local inflamma-
tion in parallel with a change in synovial histology compris-
ing a reduction in the lymphocyte number. However, we
did not observe changes in the number of macrophages, as
previously reported following 2 weeks of treatment with
daily high-dose oral corticosteroids (26). The difference in
results between the 2 studies may be due to the use of
different dosages as well as to the use of different routes of
administration of the corticosteroids, and possibly also to
the less restrictive selection criteria in our present study
that allowed inclusion of patients with both RA and
non-RA inflammatory arthritides. Nevertheless, it is inter-
esting to point out that we observed a clear clinical benefit
paralleled by a change of synovial histology that did not
include the macrophage population.
Since previous studies indicated that the anti-
inflammatory effect of corticosteroids might contribute
to slowing of damage progression in the injected joints,
we evaluated the effect of corticosteroids on the
RANKL/OPG system, considered a main determinant
of bone biology in different types of inflammatory
arthritis (27). To date, there are few reports of studies
regarding the in vivo influence of effective antirheumatic
drugs on the RANKL/OPG axis. We have previously
demonstrated that therapy with both infliximab and
etanercept is able to decrease the RANKL:OPG ratio
through up-regulation of synovial OPG expression (28).
We demonstrate here that corticosteroids, in contrast to
TNF antagonists, modulate the synovial RANKL/OPG
system through down-regulation of RANKL. Taken
together, our findings offer a rationale for combination
therapy with TNF antagonists and intraarticular cortico-
steroids with the aim of a dual positive modulation of the
RANKL/OPG system, resulting in bone protection.
Initial studies regarding RANKL cellular local-
ization in RA synovial tissue identified CD3? T cells
as the source of RANKL messenger RNA (29) and
protein expression (30). In contrast to these observa-
tions, a subsequent study identified RANKL expression
in approximately half of the CD68? synovial popula-
tion (31). The results of our study are consistent with a
T cell–restricted expression pattern of RANKL and an
absence of macrophage expression as evident by double-
staining with CD163. We chose to use CD163 to identify
synovial macrophages due to its higher specificity for the
macrophage lineage as compared with CD68 (32).
Besides the effect exerted by corticosteroids at
1470 MAKRYGIANNAKIS ET AL
the synovial level, we also studied potential effects
directly at the bone level by employing an in vitro
approach. In these studies, corticosteroids (DEX)
changed the phenotype of TNF-primed osteoblasts
through down-regulation of RANKL expression without
influencing OPG expression. This is an interesting find-
ing in light of the fact that TNF is considered a central
mediator for bone destruction in RA (33). We also
confirm previous data showing an increase of the
RANKL:OPG ratio in the absence of the inflammatory
stimulus. It might be that local delivery of cortico-
steroids at the site of inflammation in acute, severe
flares of disease is a valuable therapeutic choice to
combat inflammation and bone destruction without the
increased risk of osteoporosis seen with long-term sys-
temic administration of high doses. Further studies are
needed to identify the exact molecular mechanism un-
derlying the bone-protective effect of corticosteroids in
the presence of an inflammatory stimulus, but recent
findings regarding the relevance of the cyclooxygenase 2
(COX-2) pathway both in bone biology (34,35) and in
RA pathogenesis (36) might offer a clue. Corticosteroids
are able to decrease COX-2 expression in osteoblast
cultures (37) as well as in the rheumatoid synovium (38),
thus potentially modulating COX-2–dependent RANKL
The present study demonstrates that in inflam-
matory arthritis, therapy with intraarticular cortico-
steroids modulates the RANKL/OPG system toward a
bone-protective effect. We propose this mechanism as
an interesting potential molecular explanation for the
mode of action of intraarticular corticosteroids.
We thank Associate Professor Robert Harris for lin-
guistic advice and Marianne Engstrom for excellent technical
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