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Treatment with a neutralizing anti-murine interleukin-17 antibody after the onset of collagen-induced arthritis reduces joint inflammation cartilage destruction and bone erosion.

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ARTHRITIS & RHEUMATISM
Vol. 50, No. 2, February 2004, pp 650–659
DOI 10.1002/art.20001
© 2004, American College of Rheumatology
Treatment With a Neutralizing Anti-Murine Interleukin-17
Antibody After the Onset of Collagen-Induced Arthritis
Reduces Joint Inflammation, Cartilage Destruction,
and Bone Erosion
Erik Lubberts,1 Marije I. Koenders,1 Birgitte Oppers-Walgreen,1 Liduine van den Bersselaar,1
Christina J. J. Coenen-de Roo,2 Leo A. B. Joosten,1 and Wim B. van den Berg1
Objective. Interleukin-17 (IL-17) is a proinflammatory cytokine that is expressed in the synovium of
rheumatoid arthritis (RA) patients. This T cell cytokine
is implicated in the initiation phase of arthritis. However, the role of IL-17 during the effector phase of
arthritis has still not been identified; this was the
objective of the present study.
Methods. Mice with collagen-induced arthritis
(CIA) were treated with polyclonal rabbit anti-murine
IL-17 (anti–IL-17) antibody–positive serum or normal
rabbit serum after the first signs of arthritis. In addition, during a later stage of CIA mice were selected and
treated with anti–IL-17 antibody or control serum.
Arthritis was monitored visually, and joint pathology
was examined radiologically and histologically. Systemic IL-6 levels were measured by enzyme-linked immunosorbent assay, and local synovial IL-1 and receptor activator of NF-␬B ligand (RANKL) expression was
analyzed using specific immunohistochemistry.
Results. Treatment with a neutralizing anti–IL-17
antibody after the onset of CIA significantly reduced the
severity of CIA. Radiographic analysis revealed marked
suppression of joint damage in the knee and ankle
joints. Histologic analysis confirmed the suppression of
joint inflammation and showed prevention of cartilage
and bone destruction after anti–IL-17 antibody therapy.
Systemic IL-6 levels were significantly reduced after
anti–IL-17 antibody treatment. Moreover, fewer IL-1␤–
positive and RANKL-positive cells were detected in the
synovium after treatment with neutralizing IL-17. Interestingly, initiation of anti–IL-17 antibody therapy during a later stage of CIA, using mice with higher clinical
arthritis scores, still significantly slowed the progression of the disease.
Conclusion. IL-17 plays a role in early stages of
arthritis, but also later during disease progression.
Systemic IL-6 was reduced and fewer synovial IL-1–
positive and RANKL-positive cells were detected after
neutralizing endogenous IL-17 treatment, suggesting
both IL-1–dependent and IL-1–independent mechanisms of action. Our data strongly indicate that IL-17
neutralization could provide an additional therapeutic
strategy for RA, particularly in situations in which elevated IL-17 may attenuate the response to anti–tumor
necrosis factor/anti–IL-1 therapy.
Supported by a grant from the Dutch Arthritis Association
(NR-00-1-302). Dr. Lubberts’ work was supported by a Veni Fellowship of the Netherlands Organization for Scientific Research (NWO
grant 906-02-038).
1
Erik Lubberts, PhD, Marije I. Koenders, MSc, Birgitte
Oppers-Walgreen, Liduine van den Bersselaar, Leo A. B. Joosten,
PhD, Wim B. van den Berg, PhD: University Medical Center Nijmegen, Nijmegen, The Netherlands; 2Christina J. J. Coenen-de Roo,
MSc: NV Organon, Oss, The Netherlands.
Address correspondence and reprint requests to Erik Lubberts, University Medical Center Nijmegen, Nijmegen Center for
Molecular Life Sciences, Rheumatology and Advanced Therapeutics, 189, 6500 HB Nijmegen, The Netherlands. E-mail:
E.Lubberts@reuma.umcn.nl.
Submitted for publication February 26, 2003; accepted in
revised form October 21, 2003.
Interleukin-17 (IL-17) is a T cell–derived cytokine produced by activated T cells, predominantly activated CD4⫹,CD45RO⫹ memory T cells (1,2). This
cytokine may play a role in T cell–triggered inflammation by stimulating stromal cells to secrete various
cytokines and growth factors associated with inflammation (1–4). A pathogenic role for IL-17 was found in
organ allograft rejection (5), and increased IL-17 expression was detected in several diseases, such as systemic
sclerosis (6), nephrotic syndrome (7), systemic lupus
650
ANTI–IL-17 THERAPY AFTER ONSET OF CIA
erythematosus (8), and rheumatoid arthritis (RA)
(9,10). In contrast with the restricted expression of
IL-17, the IL-17 receptor is ubiquitously expressed in
virtually all cells and tissues. It is a type I transmembrane
protein that has no sequence similarity with any other
known cytokine receptor (3). Binding of IL-17 to its
unique receptor results in activation of the adaptor
molecule tumor necrosis factor (TNF) receptor–
associated factor 6, which is required for IL-17 signaling
(11).
RA is considered a systemic Th1-associated inflammatory joint disease that is characterized by chronic
synovitis and destruction of cartilage and bone. T cells
represent a large proportion of the inflammatory cells
invading the synovial tissue. Since the etiology of RA is
still unknown, regulating the cytokine imbalance might
represent an effective way to control this disease. The
proinflammatory cytokines TNF␣ and IL-1␤ play a
crucial role in the pathology of arthritis, driving enhanced production of cytokines, chemokines, and degradative enzymes (12). In vivo studies have shown that
neutralizing TNF␣ or IL-1␤ controls chronic inflammation and cartilage degradation, respectively (13–15).
Consistent with this, clinical studies revealed efficacy
after blocking of TNF␣ or IL-1␤. However, a subset of
patients did not respond to these inhibitors, and none of
the treatments cured the disease. Therefore, it is tempting to speculate that cytokines or factors other than
IL-1␤ and TNF␣ also participate in the proinflammatory
cytokine cascade.
T cell cytokine IL-17 is spontaneously produced
by RA synovial membrane cultures (9), and high levels
have been detected in the synovial fluid of patients with
RA (9,10). IL-17 can stimulate the production of IL-1␤
and TNF␣ from macrophages (4) and triggers human
synoviocytes to produce IL-6, IL-8, granulocyte–
macrophage colony-stimulating factor, and prostaglandin E2 (2,16), suggesting that IL-17 could be an upstream mediator in the pathogenesis of arthritis. Early
neutralization of endogenous IL-17 prior to the development of arthritis in the experimental arthritis model
suppresses the onset of disease (17,18). Furthermore,
IL-17 may be involved in tissue destruction. IL-17 has
biologic activities similar to those of IL-1␤, and additive/
synergistic effects with IL-1␤ and TNF␣ have been
reported (19). In vitro, IL-17 suppresses matrix synthesis
by articular chondrocytes through enhancement of nitric
oxide (NO) production (20,21). In addition, in vitro
studies suggested a role for IL-17 in bone erosion by
induction of receptor activator of NF-␬B ligand
(RANKL) expression (22). Recently, we showed that
651
IL-17 promotes bone erosion in murine collageninduced arthritis (CIA) through loss of the RANKL/
osteoprotegerin (OPG) balance (23). These observations indicate that IL-17 may promote joint
inflammation as well as tissue destruction during the
initial phase of arthritis. However, the role of T cell
IL-17 during the effector phase of arthritis has still not
been identified.
In the present study, we demonstrated the therapeutic effect of anti–IL-17 antibody treatment in CIA,
implying that the T cell cytokine IL-17 not only plays a
role in the early stage of arthritis, but also has a function
in propagating and prolonging the arthritis. Furthermore, fewer synovial IL-1␤–positive and RANKLpositive cells were found after treatment with neutralizing endogenous IL-17. This suggests that IL-17 might be
a novel target for the treatment of destructive arthritis
and implies that neutralization of this T cell factor
during the effector phase of arthritis has therapeutic
potential. Our data suggest that anti–IL-17 cytokine
therapy is an interesting new approach that may contribute to the prevention of joint destruction and could
provide an important additional strategy to the current
anti-TNF and anti–IL-1␤ therapy for RA.
MATERIALS AND METHODS
Animals. Male DBA-1/Bom mice were purchased from
Bomholtgård (Ry, Denmark). The mice were housed in filtertop cages. Arthritis was induced in mice between 10 and 12
weeks of age. Water and food were provided ad libitum.
Animal studies were approved by the Institutional Review
Board.
Induction of CIA. Bovine type II collagen (CII) was
prepared as previously described (24) and diluted in 0.05M
acetic acid to a concentration of 2 mg/ml. This was emulsified
in equal volumes of Freund’s complete adjuvant (CFA) (2
mg/ml Mycobacterium tuberculosis, strain H37Ra; Difco, Detroit, MI). DBA-1/Bom mice were immunized at the base of
the tail with 100 ␮g of bovine CII. On day 21, mice received an
intraperitoneal (IP) booster injection of 100 ␮g of CII dissolved in phosphate buffered saline (PBS), and the onset of
arthritis usually occurred a few days after the booster injection.
Assessment of arthritis. Mice were considered to have
arthritis when significant changes in redness and/or swelling
were noted in the digits or in other parts of the paws. Knee
joint inflammation was scored visually after skin dissection,
using a scale of 0–2 where 0 ⫽ uninflamed, 1 ⫽ mild, 1.5 ⫽
marked, and 2 ⫽ severe. Scoring was done by 2 independent
observers (EL, MIK) without knowledge of the experimental
groups.
Rabbit anti-murine IL-17 antisera. Polyclonal rabbit
antibodies were raised against recombinant murine IL-17
(mIL-17; R&D Systems, Minneapolis, MN) in our laboratory
by immunization with CFA, and repeated subcutaneous injec-
652
tion of IL-17 mixed with Alum (Pierce, Rockford, IL). Anti–
IL-17 titers were determined by a specific mIL-17 enzymelinked immunosorbent assay (ELISA; R&D Systems). All sera
were complement deactivated. Anti–IL-17–positive sera
blocked the stimulatory capacity of recombinant mIL-17 to
induce NO production in the murine chondrocyte cell line H4
(25). Using this in vitro system, no cross-reactivity with IL-1␤,
IL-1␣, or TNF␣ (R&D Systems) was observed.
Study protocol. CIA was induced in male DBA-1/Bom
mice as described above. After the first clinical signs of arthritis
were observed (clinical arthritis score between 0.25 and 0.75),
a single injection with the polyclonal mIL-17 antiserum (200
␮l/mouse) was given. Furthermore, mice with a higher CIA
score (clinical arthritis score between 1.0 and 1.5) were selected and treated with a single IP injection of the polyclonal
anti–mIL-17 serum (200 ␮l/mouse). As a control, the same
amount of normal rabbit serum was injected. The appearance
of arthritis in the joints was assessed and the severity score was
recorded as previously described (24). Thereafter, knee and
ankle joints were isolated and processed by light microscopy.
Assessment of the specificity of anti–IL-17 antibody in
vitro. The H4 chondrocyte cell line was used to examine the
specificity of the anti–IL-17 antibody. H4 cells (105) were
incubated with IL-17 (25 ng/ml), IL-1␣ (10 ng/ml), IL-1␤ (1
ng/ml), or TNF␣ (10 ng/ml) with or without anti–IL-17 antibody (1:400) using RPMI 1640 tissue culture medium supplemented with 0.1% bovine serum albumin (BSA). After 24
hours, 100 ␮l of conditioned medium was mixed with 100 ␮l of
Griess reagent (0.1% N-[1-naphthyl]ethylenediamine dihydrochloride [Sigma, St. Louis, MO] in 5% H3PO4) in a flatbottomed microtiter plate (Costar, Cambridge, MA), and the
optical density at 545 nm (OD545) was measured using an
ELISA plate reader (Titertek Multiscan MCC 340; Labsystems, Helsinki, Finland).
Histologic analysis. Mice were killed by cervical dislocation. Thereafter, whole knee and/or ankle joints were removed and fixed for 4 days in 10% formalin. After decalcification in 5% formic acid, the specimens were processed for
paraffin embedding (26). Tissue sections (7 ␮m) were stained
with hematoxylin and eosin or Safranin O. Histopathologic
changes were scored using the following parameters. Infiltration of cells was scored on a scale of 0–3, depending on the
amount of inflammatory cells in the synovial cavity (exudate)
and synovial tissue (infiltrate). Proteoglycan depletion was
determined using Safranin O staining. The loss of proteoglycans was scored on a scale of 0–3, ranging from fully stained
cartilage to destained cartilage or complete loss of articular
cartilage.
A characteristic parameter in CIA is the progressive
loss of articular cartilage. This destruction was graded separately on a scale of 0–3, ranging from the appearance of dead
chondrocytes (empty lacunae) to the complete loss of articular
cartilage (cartilage surface erosion). The degree of chondrocyte death was scored on a scale of 0–3, ranging from no empty
lacunae to complete loss of chondrocytes in the cartilage layer.
Cartilage surface erosion was scored on a scale of 0–3, ranging
from no cartilage loss to complete loss of articular cartilage.
Bone destruction was graded on a scale of 0–5, ranging from
no damage to the complete loss of bone structure. Histopathologic changes in the ankle joints were scored on 5 semiserial
sections of the joint, spaced 70 ␮m apart. Two observers (EL,
LUBBERTS ET AL
MIK) without knowledge of the experimental group, as described earlier (24), performed scoring.
Immunohistochemistry for RANKL and IL-1␤. Whole
ankle joints were fixed, decalcified, and embedded in paraffin,
as described above. Tissue sections (7 ␮M) were treated with
3% H2O2 for 10 minutes at room temperature. Sections were
incubated for 2 hours with 10 mM citrate (pH 6.0), and
thereafter were incubated for 1 hour with the primary antibody
directed against RANKL (rabbit polyclonal antibody raised
against the epitope corresponding to amino acids 46–317 of
RANKL of human origin [FL-317]) or IL-1␤ (rabbit antimouse IL-1␤ antibody [H153]) (Santa Cruz Biotechnology,
Santa Cruz, CA) (27). Rabbit IgG antibody (X0936; Dako,
Carpinteria, CA) was used as a control. After rinsing, sections
were incubated for 30 minutes with biotinylated horseradish
peroxidase–conjugated goat anti-rabbit IgG (Dako P0448).
Development of the peroxidase staining was done with diaminobenzidine (Sigma). Counterstaining was done with Mayer’s
hematoxylin.
Sections were coded and randomly analyzed by 2
independent observers (EL, BO-W). Staining of IL-1␤ and
RANKL was semiquantitatively scored on a 5-point scale
(scores 0–4) at 200⫻ magnification; a score of 0 represented
no staining and a score of 4 represented staining of a high
number of inflammatory cells. IL-1␤–positive and RANKLpositive cells were counted manually in 5 random high-power
fields, and then averaged and scored on a scale of 0–4 as
follows: 0 (no staining), 1 (1–5 positive cells), 2 (6–10 positive
cells), 3 (11–15 positive cells), and 4 (⬎20 positive cells).
Radiologic assessment. At the end of the experiment,
knee and/or ankle joints were isolated and used for radiographic analysis as a marker for joint destruction. Radiographs
were carefully examined using a stereomicroscope, and joint
destruction was scored on a scale of 0–5, ranging from no
damage to complete destruction of the joint (24).
Determination of IL-6 protein. IL-6 levels in sera were
measured by a specific ELISA. Anti-murine IL-6 antibodies
were from BioSource International (Camarillo, CA) (capture
antibody rat anti-mouse IL-6 monoclonal antibody [mAb]
[MP5-20F3], detection antibody rat anti-mouse IL-6 mAb
biotin-labeled [MP5-32CK]). No cross-reactivity with the cytokines IL-1␤, IL-10, and TNF␣ was found. Briefly, ELISA
plates were coated with the capture antibody (3 ␮g/ml) by
overnight incubation at 4°C in carbonate buffer (pH 9.6).
Nonspecific binding sites were blocked by incubation for 1
hour at 37°C with 1% BSA in PBS/Tween. The sera from mice
of different experimental groups were tested by incubation for
2 hours at room temperature. The plates were then incubated
for 2 hours at room temperature with the biotinylated second
antibody, followed by a 30-minute incubation at 37°C with
streptavidin–polyperoxidase conjugate. Bound complexes were
detected by reaction with orthophenylenediamine and H2O2.
Absorbance was measured at 492 nm by an ELISA plate
reader. The cytokine concentration in the samples was calculated as pg/ml using recombinant murine IL-6 (BioSource
International) as a standard. The sensitivity of the IL-6 ELISA
was 32 pg/ml.
Statistical analysis. Differences between experimental
groups were tested using the Mann-Whitney U test, unless
stated otherwise. The data are expressed as the mean ⫾ SEM.
ANTI–IL-17 THERAPY AFTER ONSET OF CIA
653
Figure 1. Specificity of the anti–interleukin-17 (anti–IL-17) antibody
in vitro. H4 cells were incubated with IL-17, IL-1␣, IL-1␤, or tumor
necrosis factor ␣ (TNF␣) with or without anti–IL-17 antibody, in
triplicate for 24 hours. RPMI 1640 tissue culture medium was used as
a control. Thereafter, 100 ␮l of cell culture medium was mixed with
100 ␮l of Griess reagent. The optical density (OD) was measured by an
enzyme-linked immunosorbent assay plate reader. Data are expressed
as OD545 and are the mean and SEM of 2 separate experiments.
RESULTS
Effects of neutralizing endogenous IL-17 using
anti–IL-17 antibody treatment. Polyclonal rabbit antimurine IL-17 antibody–positive serum was generated in
our laboratory. To examine the neutralizing capacity of
the anti–IL-17 antibody, H4 chondrocytes were stimulated with IL-17 with or without the anti–IL-17 antibody
for 24 hours. IL-17 stimulated H4 chondrocytes to
produce NO in the supernatant, which was measured as
OD545. H4 cells incubated with IL-17 resulted in an
increase in OD545 (Figure 1). Coincubation with the
anti–IL-17 antibody prevented the IL-17–induced increase, showing an IL-17 neutralizing capacity of the
anti–IL-17 antibody (Figure 1). To further investigate
the specificity of the anti–IL-17 antibody, similar studies
were performed in which H4 cells were incubated with
IL-1␣, IL-1␤, or TNF␣ cytokines. Coincubation with the
anti–IL-17 antibody had no effect on the increase in OD
induced by these proinflammatory cytokines (Figure 1).
This indicates that the anti–IL-17 antibody had no
cross-reactivity with these important cytokines in the
pathogenesis of arthritis.
Thereafter, arthritic mice were systemically
treated with the anti–IL-17 antibody immediately after
the first signs of CIA. As shown in Figure 2A, a single
injection with the polyclonal anti–IL-17 antibody after
the onset of CIA significantly suppressed the macro-
Figure 2. Effect of anti–interleukin-17 (anti–IL-17) antibody treatment
in collagen-induced arthritis. Immunized DBA-1/Bom mice received 1
intraperitoneal injection of anti–IL-17 antibody–positive serum after the
first signs of arthritis (clinical score between 0.25 and 0.5). As a control,
the same amount of normal rabbit serum was injected. The appearance of
arthritis was assessed, and it was scored for severity in the fore and hind
paws (A). At the end of the experiment, mice were killed by cervical
dislocation, after which the hind knee (B) and ankle (C) joints were
analyzed for joint damage by radiography. Data are the mean and SEM
of 3 separate experiments with at least 30 mice per group. ⴱ ⫽ P ⫽ 0.01;
ⴱⴱ ⫽ P ⬍ 0.005; ⴱⴱⴱ ⫽ P ⬍ 0.0005 versus control group, by MannWhitney U test.
654
LUBBERTS ET AL
control group (P ⫽ 0.04) (Figure 3A). In the control
arthritic joints, numerous granulocytes and mononuclear
cells were present and several granulocytes adhered to
cartilage. Less influx of granulocytes and mononuclear
cells was noted after anti–IL-17 antibody treatment
(Figure 4).
In addition, histologic analysis revealed a significant reduction of focal bone erosion after blocking of
IL-17 (P ⬍ 0.05) (Figures 3B and 4). Multinucleated
cells were detected at sites of focal bone erosion in the
control group (Figure 4C). However, anti–IL-17 treatment greatly decreased the number of multinucleated
cells in the joint (Figure 4D).
Prevention of chondrocyte death and cartilage
surface erosion with anti–IL-17 antibody treatment.
Joint sections were stained with Safranin O to investigate proteoglycan content in the articular cartilage.
Furthermore, semiserial sections were scored for the
degree of chondrocyte death and cartilage surface erosion. As shown in Figure 3C, marked proteoglycan
depletion was observed in the control group, with significantly less after blocking of endogenous IL-17 (P ⫽
0.04). In addition to the analysis of reversible proteoglycan loss, joint sections were scored for the degree of
Figure 3. Histologic analysis of joint inflammation, bone erosion, and
cartilage destruction after anti–interleukin-17 (anti–IL-17) cytokine
therapy. Immunized DBA-1/Bom mice received 1 intraperitoneal
injection of anti–IL-17 antibody–positive serum or normal rabbit
serum (control) after the first signs of arthritis. Ten days later, mice
were killed by cervical dislocation and the ankle joints were obtained
for histologic analysis. Synovial inflammation (A), proteoglycan depletion (C), chondrocyte death (D), and cartilage surface erosion (E) were
scored on a scale of 0–3. Focal bone erosion (B) was scored on a scale
of 0–5. Data are the mean and SEM of 2 separate experiments with at
least 9 mice per group. ⴱ ⫽ P ⬍ 0.05 versus control group, by
Mann-Whitney U test.
scopic arthritis score compared with the control group.
Radiographic analysis revealed significantly less joint
destruction in the knee (P ⫽ 0.01) and ankle (P ⬍
0.0005) after anti–IL-17 therapy compared with the
control group (Figures 2B and C).
Reduction of synovitis and prevention of focal
bone erosion with anti–IL-17 treatment. Histologic analysis revealed significant reduction of cell influx in the
joint after blocking of endogenous IL-17 activity using
the polyclonal anti–IL-17 antibody compared with the
Figure 4. Effects of anti–interleukin-17 (anti–IL-17) cytokine therapy
on synovial inflammation and joint destruction. A and C, Sections of an
ankle joint of a mouse 10 days after a single systemic injection with
normal rabbit control serum, showing pronounced inflammation,
cartilage destruction (A) (arrowheads), and bone erosion (C) (arrows).
B and D, Sections of an ankle joint of a mouse 10 days after a single
systemic injection of anti–IL-17 antibody–positive serum. Note the
decreased synovial inflammation, cartilage destruction, and bone
erosion. BM ⫽ bone marrow; B ⫽ bone; C ⫽ cartilage; S ⫽ synovitis.
(Original magnification ⫻ 200.)
ANTI–IL-17 THERAPY AFTER ONSET OF CIA
655
Figure 5. Suppression of systemic interleukin-6 (IL-6) protein level by
systemic anti–IL-17 cytokine therapy. Immunized DBA-1/Bom mice
received 1 intraperitoneal injection of anti–IL-17 antibody–positive
serum or normal rabbit serum (control) after the first signs of arthritis.
Ten days later, sera were collected and IL-6 levels were measured by
a specific enzyme-linked immunosorbent assay, as described in Materials and Methods. Data are the mean and SEM of 10 mice per group.
ⴱ ⫽ P ⬍ 0.05 versus control group, by Mann-Whitney U test.
irreversible cartilage damage. Anti–IL-17 antibody therapy resulted in a significant reduction of chondrocyte
death (P ⬍ 0.05) and cartilage surface erosion (P ⫽ 0.02)
(Figures 3D and E and Figure 4). This indicates that
neutralizing IL-17 reduced the degree of cartilage destruction.
Anti–IL-17 antibody treatment suppresses serum
IL-6 levels. To gain insight into the mechanism of action
during IL-17 neutralization, serum levels of IL-6 were
measured in the treated animals at the time they were
killed. Ten days after the onset of arthritis, anti–IL-17
antibody treatment significantly reduced the serum levels of IL-6 (74% lower [P ⫽ 0.02] compared with the
control group) (Figure 5).
Reduced number of synovial RANKL- and IL1␤–positive cells after anti–IL-17 antibody treatment.
Specific immunohistochemistry revealed fewer IL-1␤–
positive and RANKL-positive cells in the synovium of
mice treated with the polyclonal anti–IL-17 antibody
compared with the control group (P ⬍ 0.05) (Figure 6).
This indicates that the joint-protective effect after neutralizing endogenous IL-17 might be mediated by suppression of synovial IL-1␤ and RANKL production.
Role of T cell IL-17 in prolongation of CIA. The
role of T cell IL-17 during joint inflammation and tissue
destruction at a later stage of CIA is unknown and may
be limited and overruled by monocyte/macrophage ac-
Figure 6. Effects of anti–interleukin-17 (anti–IL-17) treatment on
IL-1␤ and receptor activator of NF-␬B ligand (RANKL) expression in
the synovium. Specific immunohistochemistry revealed A, IL-1␤ and
B, RANKL-positive cells in the synovium of the control group.
Neutralizing IL-17 resulted in a significant reduction of synovial
IL-1␤–positive and RANKL-positive cells. Data are the mean and
SEM of 2 separate experiments with at least 9 mice per group. See
Figure 2 for more detail on the experimental protocol. No staining was
observed in serial sections of the same area using the rabbit IgG
control antibody (results not shown). ⴱ ⫽ P ⬍ 0.05 versus control
group, by Mann-Whitney U test.
tivity. Therefore, we examined whether T cell IL-17
plays a role in the prolongation of CIA. Anti–IL-17
antibody therapy was started during a later stage of CIA
using mice with a clinical CIA score in the ankle joints
between 1 and 1.5. As shown in Figure 7, a single
systemic injection with the polyclonal anti–IL-17 antibody significantly slowed the progression of the disease
in the ankle and knee joints, indicating that T cell IL-17
plays a role in the prolongation of CIA.
656
LUBBERTS ET AL
Figure 7. Role of T cell interleukin-17 (IL-17) in prolongation of
collagen-induced arthritis. Arthritic mice with a clinical score in the
ankle joints between 1 and 1.5 received 1 intraperitoneal injection of
anti–IL-17 antibody–positive serum. As a control, the same amount of
normal rabbit serum was injected. The appearance of arthritis was
assessed, and it was scored for severity in the fore and hind paws (A)
and hind knee joints (B). There was significant suppression of the
disease progression in the fore and hind paws and knee joints after
neutralizing endogenous IL-17. Data are the mean and SEM of at least
10 mice per group. ⴱ ⫽ P ⫽ 0.03; ⴱⴱ ⫽ P ⫽ 0.009 versus control group,
by Mann-Whitney U test.
DISCUSSION
This is the first study to demonstrate that neutralizing T cell IL-17 during the effector phase of CIA
has therapeutic potential. Radiologic and histologic analyses revealed significant protection from joint damage
when endogenous IL-17 was neutralized after arthritis
expression. This protective effect was associated with
down-regulation of IL-1␤ and RANKL in the synovium.
Furthermore, neutralizing endogenous IL-17 at a later
stage of CIA still slowed the progression of the disease.
IL-17 shares many properties with IL-1␤ and
TNF␣, and this T cell–derived cytokine has been shown
to be present in the synovium of RA patients (9,10).
IL-17 induces the production of proinflammatory mediators, such as IL-1␤ and TNF␣, from several joint cells
including synovial fibroblasts, macrophages, and chondrocytes. In addition, IL-17 induces RANKL expression
(22,23), which is a novel cytokine crucial for osteoclastogenesis (28). Moreover, IL-17, IL-1␤, TNF␣, and
RANKL activate the common transcription factor
NF-␬B in a variety of cells. IL-17 can synergize with
these cytokines (IL-1␤, TNF␣, and RANKL), but probably has direct activity as well (17,29). Since a subset of
RA patients does not respond to neutralizing IL-1␤ or
TNF␣, other factors, such as IL-17, may participate in
the proinflammatory cytokine cascade. In the present
study, we showed the involvement of IL-17 in joint
inflammation and prolongation of arthritis.
IL-17 has dual effects on cartilage. In vitro, it
inhibits chondrocyte metabolism in intact articular cartilage of mice and results in proteoglycan breakdown
(19,20,30,31). Furthermore, in vitro studies show the
induction of metalloproteinases by IL-17 in synoviocytes
and chondrocytes (32–34). Interestingly, the effects of
IL-17 on matrix degradation and synthesis were not
dependent on IL-1␤ production by chondrocytes, and
IL-1 receptor antagonist did not block IL-17–induced
matrix release nor did it prevent the inhibition of matrix
synthesis in vitro using porcine articular cartilage explants (31). Moreover, we recently identified an IL-1␤–
independent role of IL-17 in the pathogenesis of arthritis in vivo (17). The downstream signaling pathways for
IL-17 and IL-1␤ seem to be distinct, and differential
activation of activator protein 1 members by IL-17 and
IL-1␤ has been described (33). IL-1␤ is by far the more
catabolic cytokine in experimental arthritis compared
with TNF␣; however, IL-17 synergizes with TNF␣ to
induce cartilage destruction in vitro (35). This underscores the potential of IL-17 to act additively or even
synergistically with IL-1␤/TNF, but IL-17 may have
direct catabolic effects as well. In the present study, we
found a clear reduction of synovial IL-1␤ expression
after neutralizing endogenous IL-17 in CIA, indicating
that IL-17 is an upstream mediator of IL-1␤. Furthermore, we showed IL-17 to be a novel target for the
treatment of cartilage destruction in experimental arthritis.
IL-17 seems to be a potent stimulator of osteoclastogenesis (22,23). In the present study, we found
ANTI–IL-17 THERAPY AFTER ONSET OF CIA
reduced multinucleated cells after neutralizing endogenous IL-17, indicating that anti–IL-17 antibody treatment prevents the formation of osteoclast-like cells.
Recently, it was shown that overexpression of IL-17 in
the knee joints of CIA mice results in elevated expression of RANKL in the synovium and loss of the
RANKL/OPG balance, leading to enhanced bone resorption (23). Of interest, systemic OPG treatment
inhibits the local IL-17–induced bone resorption in the
knee joints of CIA mice, suggesting that IL-17–induced
bone erosion is at least partly mediated by RANKL (23).
The observation that neutralizing IL-17 results in fewer
RANKL-positive cells in the synovium further implies a
relation between IL-17 and RANKL expression. Furthermore, it underscores the role of IL-17 in enhancement of the joint destruction process and makes IL-17
an attractive target for the treatment of destructive
arthritis. Osteoclasts are potent bone-resorbing cells and
play a crucial role in joint destruction (36). RANKL and
TNF␣ contribute to osteoclast formation, while several
other cytokines are responsible for osteoclast survival
and/or activation. Neutralizing the RANKL/RANK
pathway by administration of OPG prevents bone destruction (37–39). However, this kind of treatment is not
antiinflammatory or chondroprotective, as shown in the
present study with anti–IL-17, suggesting anti–IL-17 as a
more appropriate therapy for destructive arthritis.
In the present study, we used an anti–IL-17
antibody to neutralize endogenous IL-17 directly after
onset and during a later stage of CIA. A single injection
with anti–IL-17 antibody seems to be much more efficient than treatment with the soluble IL-17 receptor
(sIL-17R):Fc fusion protein, as previously described
(17,18). Since the treatment protocols are not identical
between our previous study and the present study, we
also treated CIA mice with the sIL-17R:Fc fusion protein starting after the onset of CIA (4 injections on
alternative days using the same dose as described in ref.
17). This treatment did not result in suppression of the
arthritis score; however, radiographic analysis revealed a
significant reduction of the degree of joint destruction.
In addition, semiquantitative analysis of messenger
RNA (mRNA) expression for IL-1␤ and RANKL using
reverse transcriptase–polymerase chain reaction showed
down-regulation of IL-1␤ and RANKL mRNA expression in the synovium after sIL-17R:Fc treatment (Lubberts E, et al: unpublished observations). It is known
that despite the ability of IL-17 to signal at low concentrations, it shows a low affinity to its receptor, with Ka
values between 2.107 and 2.108 M⫺1 (40). Therefore, we
speculate that the difference in affinity for IL-17 be-
657
tween the IL-17R:Fc and the anti-murine IL-17 antibodies may be an important reason for the higher efficacy
using the anti–IL-17 antibody. Affinity studies must be
performed to prove this hypothesis, and this is currently
under investigation.
The role of T cell cytokines such as IL-17 in
propagating and prolonging arthritis must be identified.
T cells and their cytokines may play an important role in
initiating the arthritis and during an early phase. However, during the later stage of the arthritis, T cell
cytokines may be overruled by mediators produced by
activated macrophages. It has been shown that IL-17
plays an inflammatory role in the initial phase of experimental arthritis (17,18). The present study makes it
clear that after the first clinical signs of arthritis, neutralizing endogenous IL-17 is still of therapeutic value.
Systemic IL-6 levels were reduced and fewer synovial
IL-1␤–positive and RANKL-positive cells were detected, suggesting both IL-1␤–dependent and IL-1␤–
independent mechanisms of action. Furthermore, even
at a later stage of CIA, T cell IL-17 contributes to
prolongation of the arthritis, since blocking endogenous
IL-17 in this phase of CIA slowed the progression of the
disease. This suggests that despite the abundant expression of macrophage mediators, which are partly produced independently of IL-17, T cell IL-17 plays a role in
maintaining the inflammation.
We speculate that neutralizing IL-17 during this
later stage of CIA has a suppressive effect on proinflammatory cytokine production. In addition, fewer additive/
synergistic effects between IL-17 and other proinflammatory cytokines such as TNF␣, IL-1␤, and IL-6 can be
expected. Previous blocking studies with anti–IL-1␤ and
anti-TNF␣ performed in our laboratory have shown that
TNF␣ plays an important role in early CIA, and IL-1␤ is
important in early and established CIA (15). IL-17 is a
potent inducer of IL-1␤ (17), and we hypothesize that
neutralizing IL-17 results in a reduction of IL-1␤ expression in the synovium during this later stage of CIA. Since
IL-1␤ expression is not completely dependent on IL-17,
not all IL-1 ␤ will be blocked, and this IL-17–
independent IL-1␤ production will contribute to the
progression of CIA. Fewer additive/synergistic effects
between IL-17 and IL-1␤ may also play a role in slowing
the progression of the disease. TNF␣ is hardly detectable in later stages of CIA; however, synergistic effects
between TNF␣ and IL-17 have been documented (35).
Blocking of IL-17 using the soluble receptor for IL-17
further improves the neutralizing effect of TNF␣ blocking after the onset of CIA (Lubberts E, et al: unpublished observations).
658
LUBBERTS ET AL
We hypothesize that the mechanisms responsible
for slowing the progression of the disease after neutralizing IL-17 during the later stage of CIA are suppression
of proinflammatory cytokines such as IL-1␤, TNF␣, and
IL-6, and elimination or reduction of the additive/
synergistic effects between IL-17 and these proinflammatory cytokines. Studies are currently being done in
our laboratory to further prove this hypothesis.
In summary, we have demonstrated the therapeutic potential of neutralizing T cell IL-17 during the
effector phase of CIA. IL-17 seems to play a role in
prolonging the arthritis process and may be considered
to be an important target for the treatment of destructive arthritis. IL-17 induced key catabolic cytokines such
as IL-1␤ and RANKL. Since it is known that this T cell
factor can have synergistic effects with catabolic/
inflammatory mediators (29), it is tempting to speculate
that IL-17 levels can make the difference in whether an
RA patient will respond to anti–TNF␣/anti–IL-1␤ therapy. Our data strongly suggest that anti–IL-17 cytokine
therapy is an interesting new antirheumatic approach
that will contribute to the prevention of joint destruction. Furthermore, neutralizing IL-17 could provide an
additional therapeutic strategy for RA, particularly in
situations where elevated levels of IL-17 may attenuate
the response of a patient to anti-TNF␣/anti–IL-1␤ therapy.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
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