| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
*
Rheumatology Research Laboratory, Department of Rheumatology, University Medical Center St. Radboud, Nijmegen, The Netherlands;
Department of Pharmacology, NV Organon, Oss, The Netherlands; and
Department of Medicine, Louisiana State University Health Sciences Center, New Orleans, LA 70112
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
protein were found in synovial tissue. Intriguingly, blocking of
IL-1
with neutralizing Abs had no effect on the IL-17-induced
inflammation and joint damage in the knee joint, implying an IL-1
independent pathway. This direct potency of IL-17 was underscored in
the unabated IL-17-induced exaggeration of bacterial cell
wall-induced arthritis in IL-1-/- mice. In conclusion,
this data shows that IL-17 contributes to joint destruction and
identifies an IL-1-independent role of IL-17. These findings suggest
IL-17 to be a novel target for the treatment of destructive arthritis
and may have implications for tissue destruction in other autoimmune
diseases. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
IL-17 may play an upstream role in T cell-triggered inflammation by
stimulating stromal cells to secrete other cytokines and growth
factors. It has the capacity to induce IL-6, IL-8, G-CSF,
PGE2, and the proinflammatory cytokines TNF-
and IL-1 (1, 5). This is compatible with the activation
of NF-
B, which is known to regulate a number of gene products
involved in cell activation and cell growth. Recently, it was found
that TNFR-associated factor-6 was required in IL-17 signal transduction
(6). A pathogenic role of IL-17 was found in organ
allograft rejection, where it promotes maturation of dendritic cell
progenitors and T cell proliferation (7). A further
proinflammatory activity of IL-17 is demonstrated by its capacity to
induce neutrophil recruitment through chemokine release
(8) and stimulation of granulopoiesis
(9).
Rheumatoid arthritis
(RA)3
is considered an autoimmune disease. It is a chronic systemic disorder
of unknown etiology. The pathogenesis of RA is still unknown and seems
to be multifactorial. Targeting the cytokine disbalance might represent
a solid way to control this disease. RA is characterized by chronic
inflammation of multiple joints and concomitant destruction of
cartilage and bone. In the synovial membrane of the joints, cell-cell
interactions between Th1 lymphocytes, monocytes, and synoviocytes
underlie the enhanced production of the pivotal proinflammatory
cytokines TNF and IL-1 (10). IL-17 production has been
demonstrated in RA synovial tissue (11), and IL-17
enhances IL-1 mediated IL-6 production in vitro (12). IL-1
is a pivotal cytokine in cartilage destruction, and IL-17 shares these
properties with IL-1, suggesting that infiltrated IL-17-producing Th1
cells may contribute to cartilage damage. In vitro studies identified
that IL-17 suppresses matrix synthesis by articular chondrocytes
through enhancement of NO production (13, 14). In
addition, in vitro studies suggested a role of IL-17 in bone erosion.
IL-17 induced the expression of receptor activator of NF-B ligand
(RANKL), which is a crucial factor in bone resorption (15, 16). These in vitro observations indicate that IL-17 may promote
both joint inflammation as well as tissue destruction.
Murine collagen-induced arthritis (CIA) is a widely accepted arthritis
model based on the generation of T cell and Ab-mediated autoimmune
reactivity against a cartilage autoantigen, type II collagen (CII). The
onset of arthritis is dependent on TNF-, whereas IL-1 is crucial
both in onset and propagation of arthritis (17). Th1 cells
play a pivotal role in this model (18). The expression can
be promoted by IL-12 administration during immunization as well as at
the time of onset of arthritis. In addition, onset of arthritis could
be blocked with anti-IL-12 Abs (19). In contrast,
arthritis expression is under the control of IL-10 and can be
suppressed by administration of the Th2 cytokines IL-4 and IL-10
(20). Recently, a major role of IL-15 was identified in
CIA (21). This cytokine is a potent inducer of IL-17
(22).
So far, no studies have been done to identify the role of IL-17 in
experimental arthritis models. In the present study, we examined the
role of endogenous IL-17 in CIA with soluble IL-17 receptor protein
(sIL-17R:Fc) and explored the amplifying activity of IL-17 by local
overexpression of IL-17 in the joint. It was found that blocking of
IL-17 significantly reduced CIA, including a clear suppression of joint
damage. In contrast, local IL-17 overexpression promoted destructive
arthritis. Of great interest, although IL-17 induced elevated levels of
IL-1, neutralization of IL-1 had no effect on this exaggeration,
identifying an IL-1 independent role of IL-17. Our results suggest that
IL-17-producing T cells can amplify arthritis, making it more
destructive, and imply that similar processes may underlie tissue
destruction in other autoimmune disorders.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Male DBA-1/BOM mice were purchased from Bomholdgärd (Ry,
Denmark). A breeder pair of IL-1-deficient mice was obtained from H.
Zheng (Merck, Rahway, NJ) (23). As control animals,
N/N (mixed 129sv x C57BL/6J background) were used. These animals
were bred at our university breeding facilities in Nijmegen, The
Netherlands. The mice were housed in filter-top cages. The mice were
used between 10 and 12 wk of age. Water and food were provided ad
libitum.
Materials
CFA and Mycobacterium tuberculosis (strain H37Ra)
were obtained from Difco, Detroit, MI. Bovine CII was prepared as
described previously (17). S. D. Lyman (Immunex,
Seattle, WA) kindly provided murine IL-17R/human IgG1 Fc fusion protein
(muIL-17R:Fc). RPMI 1640 was obtained from Life Technologies (Breda,
The Netherlands). ELISA plates (Maxisorb) were purchased from Nunc
(Copenhagen, Denmark). The following mAbs were used in the cytokine
ELISAs: rat anti-murine IL-17 Abs (capture: MAB721) and a
biotinylated goat anti-mouse IL-17 Ab (detection: BAF421) were
purchased from R&D Systems (Minneapolis, MN).
Streptavidin-polyperoxidase conjugate was obtained from CLB (Amsterdam,
The Netherlands). Recombinant muIL-17 was obtained from R&D Systems.
Rabbit anti-murine IL-1, polyclonal Abs were prepared in our
own laboratory (24).
Adenoviral vectors
AdCMVmIL-17 (AdIL-17) was constructed as reported previously (9). Briefly, pACCmIL-17 vector was cotransfected into 911 cells with XbaI-restricted AdCMVLacZ DNA by calcium-phosphate precipitation. AdIL-17 clones were screened by PCR, and protein production was confirmed by a muIL-17 bioassay (9). All lots of recombinant adenovirus (Ad) contained less than 1 endotoxin U/ml as measured by the Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD). The recombinant replication-deficient adenovirus AdCMVLuc (AdControl) was used as a control vector throughout the study. All virus preparations had a PFU-particle ratio of <100:1 (9). A total of 107 PFU of IL-17 virus and control virus represents 6.8 x 108 and 8.9 x 108 viral particles, respectively.
Induction of CIA
Bovine CII was diluted in 0.05 M acetic acid to a concentration of 2 mg/ml and was emulsified in equal volumes of CFA (2 mg/ml of M. tuberculosis). The mice were immunized intradermally at the base of the tail with 100 µl of emulsion (100 µg of collagen). On day 21, mice were given an i.p. booster injection of 100 µg of CII dissolved in PBS, and normally first signs of arthritis onset occurs around day 25–28, in particular in the ankles.
Study protocol
CIA was induced in male DBA-1 mice as described above. Just before expected onset of CIA (day 25), mice were scored visually for the appearance of arthritis. Mice without macroscopic signs of arthritis in the paws were selected. To investigate the endogenous role of IL-17, mice were i.p. injected at alternated days (total of four injections) from day 25, with different doses of muIL-17R:Fc or BSA. Mice were macroscopically scored for arthritis incidence and severity at alternated days. X-ray analysis was performed at the end of the experiment. To examine potential amplifying activity of IL-17, DBA-1 mice were immunized with a lower dose of bovine CII (50 µg of collagen), as described above. Just before expected onset of CIA (day 25), mice were anesthetized with ether and a small aperture in the skin of the knee was performed for the intraarticular (i.a.) injection procedure. When absence of arthritis was confirmed in the knee joint, i.a. injections were performed with 107 PFU per 6 µl of either an IL-17 expressing (AdIL-17) or a control vector (AdControl). Previously, we showed that this dose of adenoviral vector did not induce any inflammatory response after i.a. injection in the mouse knee joint (25). Five or 10 days after the i.a. injection of the viral vector, mice were sacrificed by cervical dislocation and the skin of the knee joint was removed. The appearance of arthritis in the injected joints was assessed and severity score was recorded as described previously (26). Thereafter, knee joints were isolated and processed for light microscopy.
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 noninflamed (0), mild (1), marked (1.5), or severe (2) inflammation. Scoring was performed by two independent observers without knowledge of the experimental groups.
Radiology
At the end of the experiment, ankle and knee joints were isolated and used for x-ray analysis as a marker for joint destruction. X-ray photographs were carefully examined with a stereo microscope, and joint destruction was scored on a scale from 0 to 5, ranging from no damage to complete destruction of the joint (27).
Determination of IL-17, TNF-, IL-1, and IL-1 protein
To determine the levels of TNF-, IL-1, and IL-1 in
washouts of joint tissue, patellae with adjacent synovium were isolated
in a standardized manner from knee joints as described previously
(13). Patella with adjacent synovium was incubated in RPMI
1640 medium with 0.1% BSA, gentamicin (50 µg/ml), and
L-glutamine (2 mM) (200 µl/patella) for 1 h at room
temperature. After supernatant was harvested, the cytokine levels of
muIL-1, muIL-1, and muTNF- were measured by RIA
(28). The sensitivity of the RIA IL-1, IL-1, and
TNF- is 20, 20, and 40 pg/ml, respectively. The cytokine levels of
muIL-17 was measured by ELISA. Briefly, ELISA plates were coated with
the capture Ab (3 µg/ml) by overnight incubation at 4°C in
carbonate buffer (pH 9, 6). Nonspecific binding sites were blocked by
1 h of incubation at 37°C with 1% BSA in PBS-Tween. The
supernatants from the patella cultures were tested by 3 h of
incubation at 37°C. The plates then were incubated for 1.5 h at
37°C with the biotinylated second Ab followed by a 30-min incubation
at 37°C with streptavidin-polyperoxidase conjugate. Bound complexes
were detected by reaction with orthophenylenediamine and
H2O2. Absorbance was
measured at 492 nm with an ELISA plate reader (Titertek Multiscan
MCC/340; Labsystems, Helsinki, Finland). The cytokine
concentration in the samples was calculated as pg/ml with recombinant
muIL-17 as a standard. The sensitivity of the IL-17 ELISA is 50 pg/ml.
To measure muIL-17 levels <50 pg/ml, a commercial muIL-17 ELISA kit
was used (Quantikine M; R&D Systems). The sensitivity of this ELISA is
<5 pg/ml.
Determination of anti-collagen Abs
IgG1 and IgG2a anti-collagen Abs titers against bovine CII were determined by an ELISA. Briefly, plates were coated with 10 µg of bovine CII, and thereafter nonspecific binding sites were blocked with 0.1 M ethanolamine (Sigma, St. Louis, MO). Serial 1:2 dilutions of the sera were added, followed by incubation with isotype-specific goat anti-mouse peroxidase (Southern Biotechnology Associates, Birmingham, AL) and substrate (5-aminosalicylic acid; Sigma). The numbers of serum samples that were analyzed varies from four to six samples per group per time point in both the endogenous IL-17 blocking experiments as well as systemic IL-17 overexpressing conditions and their control groups. Plates were read at 492 nm. Titers were expressed as the mean ± SD dilution that gave the half-maximal value.
Isolation of RNA
Mice were sacrificed by cervical dislocation, and the patella and adjacent synovium were immediately dissected (29). Synovium biopsy tissue was taken from six patella specimens. Two biopsy specimens with a diameter of 3 mm were punched out with a biopsy punch (Stifle, Wachtersbach, Germany): one from the lateral side and one from the medial side. Three lateral and three medial biopsy samples were pooled to yield four samples per group of six mice. The synovium samples were immediately frozen in liquid nitrogen. Synovium biopsy samples were ground to powder with a microdismembrator II (B. Braun, Melsungen, Germany). Total RNA was extracted in 1 ml of TRIzol reagent (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate, which is an improved single-step RNA isolation method based on the method described by Chomczynski and Sacchi (30).
PCR amplification
One microgram of synovial RNA was used for RT-PCR. Messenger RNA was reverse-transcribed to cDNA with oligo-dT primers, and one-twentieth of the cDNA was used in one PCR amplification. PCR was performed at a final concentration of 200 µM dNTPs, 0.1 µM of each primer, and 1 U of Tag polymerase (Life Technologies) in standard PCR buffer (20 mM Tris-HCl, pH 8.4, and 50 mM KCl; Life Technologies). The mixture was overlaid with mineral oil and amplified in a thermocycler (Omnigene; Hybaid, Ashford, U.K.). cDNA was amplified for 40 cycles. Message for GAPDH was amplified at 55°C with the primers described elsewhere (27). Sequence of gene-specific primers IL-17 (accession no. U43088) for RT-PCR was: 5'-mIL-17, TCT CAT CCA GCA AGA GAT CC; 3'-mIL-17, AGT TTG GGA CCC CTT TAC AC. Message for IL-17 was amplified at 60°C.
Histology
Mice were sacrificed by cervical dislocation. Thereafter, whole knee joints were removed and fixed for 4 days in 10% formalin. After decalcification in 5% formic acid, the specimens were processed for paraffin embedding (31). Tissue sections (7 µm) were stained with hematoxylin and eosin or Safranin O. Histopathological changes were scored with 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 with 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 and bone. Cartilage destruction was graded separately on a scale of 0–3, ranging from the appearance of dead chondrocytes (empty lacunae) to complete loss of the articular cartilage. Bone destruction was graded on a scale of 0–5, ranging from no damage to complete loss of the bone structure. Histopathological changes in the knee joints were scored in the patella and femur/tibia regions on five semiserial sections of the joint spaced 70 µm apart. Two observers without knowledge of the experimental group, as described earlier (27), performed scoring.
Streptococcal cell wall (SCW) preparation and induction of SCW arthritis
Streptococcus pyogenes T12 organisms were cultured
overnight in Todd-Hewitt broth. Cell walls were prepared as described
previously (32). The resulting 10,000 x g
supernatant was used throughout the experiments. These preparations
contained 11% muramic acid. Genetic background of IL-1-deficient
mice prevents induction of CIA in these mice. Therefore, unilateral
arthritis was induced in normal and IL-1-deficient mice by i.a.
injection of 25 µg of SCW (Rhamnose content) in 6 µl of PBS into
the right knee joint of naive mice. As a control, PBS was injected into
the left knee joint. To investigate the effects of local IL-17
overexpression in this model, 107 PFU of AdIL-17
or the control vector were i.a. injected in the knee joint 18 h
before inducing SCW arthritis. Four and 10 days after SCW arthritis
induction, the effects on inflammation were analyzed.
Statistical analysis
Differences between experimental groups were tested with the Mann-Whitney rank sum test, unless stated otherwise.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
To investigate IL-17 expression during early onset of CIA, DBA
mice were immunized with CII. Low levels of systemic IL-17 protein were
found over a period of 10 days after CII booster injection (range,
6–13 pg/ml). Around expected onset, synovium samples were taken at
days 26, 28, 30, and 32. Histologic analysis showed negligible and mild
inflammation at days 26 and 28 (0.1 ± 0.04 and 0.5 ± 0.4,
respectively), which was gradually increasing during time (day 30,
1.8 ± 1.2 and day 32, 2.2 ± 1.5). IL-17 expression was
examined with RT-PCR. IL-17 mRNA expression was noted at day 26 and
seems fully expressed at days 28, 30, and 32 (Fig. 1
).
|
To examine the role of IL-17, endogenous IL-17 was blocked at
expected onset of CIA. To this end, CII-immunized DBA-1 mice were given
a booster injection on day 21. At days 25, 27, 29, and 31, mice were
treated with different concentrations of soluble muIL-17 receptor
protein (muIL-17R:Fc). Blocking endogenous IL-17 dose-dependently
suppresses the arthritis incidence (Fig. 2A). At day 33, significant inhibition of the clinical score
was noted in mice treated with 25, 75, and 150 µg of muIL-17R:Fc,
although no difference in clinical score was found between the 75- and
150-µg treated groups at this time point.
|
B). Apart from
the analysis of clinical inflammation, ankle joints were taken at the
end of the experiment, and joint damage was analyzed by x-ray.
Interestingly, radiographic analysis showed less joint damage in the
muIL-17R:Fc-treated group compared with the control group
(p = 0.02; Fig. 2C), indicating a
role of endogenous IL-17 not only in inflammation but also in joint
destruction. Blocking endogenous IL-17 may influence the development of arthritis immunity. Therefore, we examined whether IL-17 plays a role in IgG1 and IgG2a anti-collagen Ab production. IgG1 and IgG2a were determined in sera at day 33. No difference in IgG1 (control, 781 ± 404 vs sIL-17R:Fc, 432 ± 255) and IgG2a (control, 714 ± 247 vs sIL-17R:Fc, 684 ± 358) production was found after blocking endogenous IL-17. Apart from measuring IgG1 and IgG2a anti-collagen Ab levels, we also analyzed the effects of blocking IL-17 in T cell responses to collagen. No differences in T cell responses to murine collagen peptides were noted between the sIL-17RFc-treated group and the control group (data not shown).
Systemic IL-17 gene transfer
To investigate the potential of IL-17 to enhance arthritis, gene
transfer with an adenoviral vector expressing IL-17 was envisaged.
CII-immunized mice were i.v. injected with 1 x
108 PFU of AdIL-17 or AdControl 20 days after
immunization. On day 21, mice were given a booster injection with CII.
At day 23, 70% arthritis incidence was already noted in mice injected
with AdIL-17 compared with 10% in the control vector group (Fig. 3A). The severity of arthritis was significantly enhanced
after systemic IL-17 gene transfer (Fig. 3B), indicating
that IL-17 accelerates CIA expression.
|
At days 24, 29, and 31, no differences were found in IgG1 and IgG2a anti-collagen Ab levels in serum between the AdIL-17 and the control vector group, although an increase in both IgG1 and IgG2a anti-collagen Ab levels was observed in both groups after CII booster injection (data not shown).
Local IL-17 gene transfer
Because systemic IL-17 gene transfer enhanced CIA and no direct
influences of IL-17 was noted in Ig production, we examined whether
local IL-17 had amplifying activity when injected in the knee joint of
CII-immunized mice. To start with, naive mice were i.a. injected in the
right knee joint with 1 x 107 PFU of
AdIL-17 or AdControl, and IL-17 levels were measured at different time
points in washouts of joint tissue. High levels were found in the first
7 days after a single injection of AdIL-17 with a maximum at day 1
(Fig. 4). No detectable levels of IL-17 were found in washouts from the
contralateral knee or the AdControl vector-injected knee (data not
shown).
|
Histologic analysis of naive mouse knee joints injected with
1 x 107 PFU of AdIL-17 revealed joint
inflammation at days 2, 5, and 10, which gradually increased during
time (Table I). Predominantly, influx of PMNs was noted at day 2, and at days 5 and
10 mononuclear cells also were found. No chondrocyte death and
cartilage erosion were observed during this time period, and only mild
bone erosion in the femur region at day 10 was found. However, local
IL-17 expression induces significant proteoglycan depletion (Table I).
Although marked influx of PMNs was apparent, these cells did not stick
to the cartilage layer (data not shown).
|
DBA-1 mice were immunized with suboptimal concentration of CII,
and shortly before expected onset of CIA, a single injection of
different doses of 105,
106, and 107 PFU of either
AdIL-17 or AdControl were i.a. injected in the right or left knee
joint, respectively. Five days after the viral injection, knee joints
were analyzed and severity of arthritis was scored. Local IL-17
overexpression during the early phase of CIA dose-dependently enhanced
the onset and promoted synovial inflammation compared with mice
injected with control vector (Table II).
|
).
|
).
Furthermore, histologic analysis showed significantly more inflammatory
cells in the synovium (infiltrate; p < 0.001) and
joint space (exudate; p < 0.001) compared with the
control vector group (Fig. 6A). In addition, we have analyzed in these experiments the
effects on the ipsilateral paw. Local IL-17 gene transfer in the knee
joint significantly increased the arthritis severity in the ipsilateral
paw (Fig. 6B).
|
In addition to inflammatory aspects, 5 days after i.a. injection
of the adenoviral vectors, chondrocyte death was observed in the
control vector group. However, local overexpression of IL-17 markedly
enhanced the degree of chondrocyte death (p <
0.001; Figs. 6A and
7). PMNs were heavily sticking to the cartilage of the patella and femur
region after IL-17 overexpression at day 5, a phenomenon hardly seen in
the control group. Scant cartilage surface erosions was noticed in the
control vector group at day 5, whereas pronounced cartilage destruction
was found in the IL-17 overexpression group (AdControl, 0.01 ±
0.1 vs AdIL-17, 0.6 ± 0.2; p < 0.001; Fig. 7).
|
and 9). Histologic analysis showed a mild degree of bone erosion in the
control group at day 5. However, pronounced bone erosion was then
already noted in the cortical bone of the patella (AdControl, 0.3
± 0.6 vs AdIL-17, 1.8 ± 0.5; p < 0.001) and
femur/tibia region (AdControl, 0.1 ± 0.2 vs AdIL-17, 0.7 ±
0.2; p < 0.001) in the IL-17 group.
|
|
in the synovium by local IL-17
In vitro studies have shown that IL-17 can stimulate the
production of IL-1 and TNF- (5, 12). Furthermore, we
have shown previously the role of IL-1 in cartilage destruction in CIA
(17, 33). Therefore, we investigated the effects of local
IL-17 overexpression on the production of the proinflammatory cytokines
IL-1 and TNF- in the synovium. As shown in Table III, local IL-17 enhances the protein expression of IL-1
(p = 0.009). No elevated levels of TNF- or
IL-1 were induced by local IL-17 (Table III). This suggests that the
local IL-17 effects can be mediated by IL-1 production in the
synovium.
|
-independent
To investigate the contribution of the proinflammatory cytokine
IL-1 on the IL-17-induced inflammation, we used specific Abs to block
IL-1 activity. Remarkably, blocking of IL-1 had no effect on
IL-17-induced acceleration of arthritis in the knee
(p < 0.001; Table IV). Moreover, x-ray analysis at day 5 revealed no difference in
IL-17-induced joint damage between the anti-IL-1 group and the
control group (1.0 ± 0.3 SEM and 1.0 ± 0.3 SEM,
respectively), although significant difference in joint damage was
found between the AdIL-17 and the control vector group at this time
point (1.0 ± 0.3 SEM and 0.1 ± 0.002 SEM, respectively;
p = 0.02). This implies that the IL-17-induced
inflammation and joint damage are independent of IL-1.
|
We further examined the IL-1-independent potential of IL-17 to
enhance joint pathology in SCW arthritis. This model was induced in
normal and IL-1-deficient mice. Interestingly, local overexpression
of IL-17 enhanced chronicity and erosive character of the SCW arthritis
in the control mice (p = 0.008). Of high
interest, similar exaggeration of arthritis was found in the
IL-1-/- mice compared with the control mice
(Fig. 10). This strongly underscores an IL-1-independent role of IL-17.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
IL-1 and TNF- are key mediators in the pathology of arthritis,
driving enhanced production of cytokines, chemokines, and degradative
enzymes (34). Neutralizing TNF- or IL-1 yielded
promising results in controlling chronic inflammation and cartilage
degradation, respectively. However, none of these 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. IL-17 has been found to stimulate the
production of IL-1 and TNF- from macrophages (5) and
triggers human synoviocytes to produce IL-6, IL-8, GM-CSF, and
PGE2 (1, 12), suggesting that IL-17
could be an upstream mediator in the pathogenesis of arthritis. In the
present study, we found amelioration of CIA after blocking of
endogenous IL-17. Furthermore, IL-17 overexpression during onset
aggravates synovial inflammation and joint destruction, a process that
was independent of IL-1. Therefore, our data put IL-17 on the list of
important proinflammatory cytokines in experimental arthritis with
destructive capacity, independent of IL-1.
RA is considered a Th1-associated disease (35). However,
the factors that initiate and sustain Th1 responses in RA synovium
still are not identified. IL-15 may replace the requirement for IL-2,
as it has been shown that it has biological functions similar to those
of IL-2. IL-15 can recruit and activate CD45RO+
memory T cell subset into the synovial membrane and can induce TNF-
production in RA (36, 37). Interestingly, these T cell
subsets are IL-17 producer cells after stimulation, and it has been
shown that IL-15 triggers IL-17 production in vitro (22).
From these studies, it is not clear whether IL-17 operates downstream
of IL-15 and whether IL-17 has a direct role in T cell activation.
Although direct T cell activation by IL-17 in vivo has not been shown,
a role for IL-17 in allogenic T cell proliferation has been suggested
(7). IL-17 can promote the development of dendritic cell
progenitors by increased surface expression of CD11c, CD40, CD80, CD86,
and MHC class II Ags. IL-17 did not significantly affect the phenotype
or function of mature dendritic cells. In the present study, no
differences in T cell responses to collagen and in IgG1 or IgG2a
anti-collagen Ab levels were found compared with the control group
after systemic overexpression of IL-17 around booster injection or
blocking endogenous IL-17 around the onset of CIA. This implies that
IL-17 did not have a role in the development of immunity against
CII.
IL-17 appears a novel target in T cell-mediated inflammatory disease,
playing a role upstream in the pathologic process. In the present
study, we found that blocking endogenous IL-17 results in suppression
of CIA, including reduction of joint damage. Apart from direct blocking
of endogenous IL-17 with soluble IL-17 receptor, previously we showed
that overexpression of IL-4 down-regulated IL-17 and IL-1 expression
and prevented cartilage and bone erosion (26, 27). In
contrast, the promotion of CIA in the knee joint by local IL-17
overexpression was not restricted to the knee joint alone. Local
overexpression of IL-17 in the knee joint also accelerates the
arthritis incidence in the ipsilateral paw. As IL-17 can increase IL-1
and TNF- production in macrophages, blocking IL-17 will also
decrease IL-1 and TNF- actions. These findings imply that regulation
of the production and activity of IL-17 may have important consequences
in the net production of proinflammatory cytokines in the synovium and
in tissue-destructive processes.
Apart from its role in the inflammatory process, IL-17 may be an
important catabolic mediator in tissue destruction. Involvement of
IL-17 in cartilage disturbance has been shown in vitro
(13), and it contributes to joint degradation in RA
(38). Furthermore, we showed that i.a. injections of
adenoviral IL-17 in the knee joint of naive mice results in
proteoglycan depletion, which is in line with the observation reported
by Dudler et al. (39). Although these studies indicate
catabolic effects of IL-17, the role of IL-1, which is the most potent
catabolic mediator in the arthritic process, cannot be excluded. Low
levels of IL-17 and IL-1 have additive or synergistic effect on joint
pathology in vitro (38). In the present study, we showed
that local overexpression of IL-17 in the knee joint of CII-immunized
mice resulted in elevated levels of IL-1 in the synovium. No
increase in TNF- and IL-1 levels was found. However, this study
did not rule out the possibility that TNF- and IL-1 were elevated
at earlier time points. It has been shown that TNF- and IL-1 play
a role especially in early CIA. IL-1 plays a dominant role in early
and late CIA. The fact that IL-17 still induced enhancement of
arthritis in the absence of IL-1 indicates an IL-1 independent role of
IL-17. Furthermore, these data suggest that IL-17 not only play a
promotive role upstream in the T cell-mediated inflammatory process but
can also function as an independent catabolic mediator. Taking this
together, IL-17 seems to be a strong IL-1-inducing cytokine in vivo and
can alone or together with IL-1 play a key role in destructive
arthritis.
In the present study, we found that local overexpression of IL-17 in the knee joint leads to enhanced influx of PMNs. This is in line with earlier reports showing that IL-17 stimulates granulopoiesis in mice (8, 9). The main difference between IL-17 overexpression in naive and CII-immunized DBA-1 mice was the observation that in CIA, PMNs were heavily sticking to patella and femur cartilage, a phenomenon that was not observed in naive mice. Interestingly, under both conditions, local IL-17 induced proteoglycan depletion. However, no chondrocyte death and cartilage erosion was observed in the naive mice after local IL-17 overexpression. In contrast, local IL-17 aggravates cartilage erosion in CIA. It has been shown that increased elastase activity was associated with neutrophil recruitment by IL-17 in airways in vivo (40). The elevated neutrophil influx and subsequent sticking to anti-CII immune complexes in the cartilage surface layer probably releases oxygen species and proteolytic enzymes present in the PMNs directly into the surface of the cartilage, thereby escaping inhibitors present in the synovial fluid (41, 42). PMNs may play an active role in cartilage destruction and need this close contact to cartilage to accomplish cartilage damage.
Here, we report that IL-17 accelerates bone erosion in CII-immunized
mice and blocking endogenous IL-17 suppresses joint damage. Activated T
cells may play a regulatory role in bone loss and joint destruction
through RANKL in vivo (43), and it has been shown that T
cell IL-17 may be a crucial cytokine for osteoclastic bone resorption
in vitro via RANKL expression (15, 44). Osteoclasts are
potent bone-resorbing cells, and RANKL has been shown to be a key
regulator of osteoclastogenesis. Moreover, RANKL binds to its receptor,
RANK, and the RANKL/RANK balance seems of crucial importance in
osteoclastogenesis and the bone erosion process. We found RANKL and
RANK protein expression in the synovium and at bone erosion sites in
the IL-17 group by specific immunohistochemistry (E. Lubberts et al.,
manuscript in preparation). The role of IL-17, in relation to
IL-1 and TNF in the regulation of RANKL and osteoclastic bone
resorption in vivo needs further clarification. In vitro, IL-17 had no
effect on basal and IL-1 stimulated osteoclastic bone resorption,
but when given together with TNF- it increased bone resorption
(44). Furthermore, TNF- can stimulate osteoclast
differentiation independent of RANKL-RANK interaction in vitro
(45). Interestingly, we found that anti-IL-1 treatment
had no effect on RANKL expression after local adenoviral IL-17
injection (E. Lubberts et al., manuscript in preparation). Further
characterization of the role of IL-17 in relation to IL-1 and TNF-
in RANKL activity is warranted.
In conclusion, this is the first report demonstrating clear contribution of IL-17 to the pathogenesis of CIA. IL-17 plays a role in joint inflammation as well as tissue destruction. Although local IL-17 enhanced IL-1 levels in the synovium, blocking IL-1 had no effect on IL-17-induced joint inflammation and joint destruction, identifying an IL-1-independent role of IL-17. Our data make it clear that IL-17 can amplify T cell-driven arthritis, making it more destructive. These findings imply that IL-17 is a novel target for destructive arthritis, and similar processes may underlie tissue destruction in other autoimmune disorders.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Erik Lubberts, University Medical Center St. Radboud, Rheumatology Research Laboratory, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. E-mail address: E.Lubberts{at}reuma.azn.nl
3 Abbreviations used in this paper: RA, rheumatoid arthritis; CIA, collagen-induced arthritis; CII, type II collagen; SCW, streptococcal cell wall; i.a., intraarticular; PMN, polymorphonuclear cell; Ad, adenovirus; mu, murine.
Received for publication November 15, 2000. Accepted for publication May 8, 2001.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
and TNF-, by human macrophages. J. Immunol. 160:3513.B together regulate interleukin-17-induced nitric oxide production in human osteoarthritic chondrocytes: possible role of transactivating factor mitogen-activated protein kinase-activated protein kinase (MAPKAPK). Arthritis Rheum. 42:2399.[Medline]
, anti-IL-1/ and IL-1Ra. Arthritis Rheum. 39:797.[Medline]
-chain administration prevents murine collagen-induced arthritis: a role for IL-15 in development of antigen-induced immunopathology. J. Immunol. 160:5654.-deficient mice. Immunity 3:9.[Medline]
(TNF), and interleukin-6 (IL-6) in cartilage proteoglycan metabolism and destruction: effect of in situ blocking in murine antigen- and zymosan-induced arthritis. Arthritis Rheum. 38:164.[Medline]
and IL-1 production. J. Appl. Physiol. 79:1497.This article has been cited by other articles:
|
|
 |
|
  I. Horie, N. Abiru, Y. Nagayama, G. Kuriya, O. Saitoh, T. Ichikawa, Y. Iwakura, and K. Eguchi T Helper Type 17 Immune Response Plays an Indispensable Role for Development of Iodine-Induced Autoimmune Thyroiditis in Nonobese Diabetic-H2h4 Mice Endocrinology, November 1, 2009; 150(11): 5135 - 5142. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. OLIVITO, G. SIMONINI, S. CIULLINI, M. MORIONDO, L. BETTI, E. GAMBINERI, L. CANTARINI, M. DE MARTINO, C. AZZARI, and R. CIMAZ Th17 Transcription Factor RORC2 Is Inversely Correlated with FOXP3 Expression in the Joints of Children with Juvenile Idiopathic Arthritis J Rheumatol, September 1, 2009; 36(9): 2017 - 2024. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Cui, H. Shao, C. Lan, H. Nian, R. L. O'Brien, W. K. Born, H. J. Kaplan, and D. Sun Major Role of {gamma}{delta} T Cells in the Generation of IL-17+ Uveitogenic T Cells J. Immunol., July 1, 2009; 183(1): 560 - 567. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Freitas, J. C. Alves-Filho, T. Victoni, T. Secher, H. P. Lemos, F. Sonego, F. Q. Cunha, and B. Ryffel IL-17 Receptor Signaling Is Required to Control Polymicrobial Sepsis J. Immunol., June 15, 2009; 182(12): 7846 - 7854. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Boldizsar, O. Tarjanyi, P. Nemeth, K. Mikecz, and T. T. Glant Th1/Th17 polarization and acquisition of an arthritogenic phenotype in arthritis-susceptible BALB/c, but not in MHC-matched, arthritis-resistant DBA/2 mice Int. Immunol., May 1, 2009; 21(5): 511 - 522. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-H. LEE, M.-L. CHO, J.-I. KIM, Y.-M. MOON, H.-J. OH, G.-T. KIM, S. RYU, S.-H. BAEK, S.-H. LEE, H.-Y. KIM, et al. Interleukin 17 (IL-17) Increases the Expression of Toll-like Receptor-2, 4, and 9 by Increasing IL-1{beta} and IL-6 Production in Autoimmune Arthritis J Rheumatol, April 1, 2009; 36(4): 684 - 692. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. S. Voo, Y.-H. Wang, F. R. Santori, C. Boggiano, Y.-H. Wang, K. Arima, L. Bover, S. Hanabuchi, J. Khalili, E. Marinova, et al. Identification of IL-17-producing FOXP3+ regulatory T cells in humans PNAS, March 24, 2009; 106(12): 4793 - 4798. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Shahrara, S. R. Pickens, A. Dorfleutner, and R. M. Pope IL-17 Induces Monocyte Migration in Rheumatoid Arthritis J. Immunol., March 15, 2009; 182(6): 3884 - 3891. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Perona-Wright, S. J. Jenkins, R. A. O'Connor, D. Zienkiewicz, H. J. McSorley, R. M. Maizels, S. M. Anderton, and A. S. MacDonald A Pivotal Role for CD40-Mediated IL-6 Production by Dendritic Cells during IL-17 Induction In Vivo J. Immunol., March 1, 2009; 182(5): 2808 - 2815. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Ke, K. Liu, G.-Q. Huang, Y. Cui, H. J. Kaplan, H. Shao, and D. Sun Anti-Inflammatory Role of IL-17 in Experimental Autoimmune Uveitis J. Immunol., March 1, 2009; 182(5): 3183 - 3190. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W Niedbala, B Cai, X Wei, A Patakas, B P Leung, I B McInnes, and F Y Liew Interleukin 27 attenuates collagen-induced arthritis Ann Rheum Dis, October 1, 2008; 67(10): 1474 - 1479. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Kochetkova, T. Trunkle, G. Callis, and D. W. Pascual Vaccination without Autoantigen Protects against Collagen II-Induced Arthritis via Immune Deviation and Regulatory T Cells J. Immunol., August 15, 2008; 181(4): 2741 - 2752. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. L. Rankin, A. J. Reed, S. Oh, C. Cozzo Picca, H. M. Guay, J. Larkin III, L. Panarey, M. K. Aitken, B. Koeberlein, P. E. Lipsky, et al. CD4+ T Cells Recognizing a Single Self-Peptide Expressed by APCs Induce Spontaneous Autoimmune Arthritis J. Immunol., January 15, 2008; 180(2): 833 - 841. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Cao, P. D. Doodes, T. T. Glant, and A. Finnegan IL-27 Induces a Th1 Immune Response and Susceptibility to Experimental Arthritis J. Immunol., January 15, 2008; 180(2): 922 - 930. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. T. Nardelli, S. M. Callister, and R. F. Schell Lyme Arthritis: Current Concepts and a Change in Paradigm Clin. Vaccine Immunol., January 1, 2008; 15(1): 21 - 34. [Full Text] [PDF]
|
|
|
|
|
|
H. W. Lim, J. Lee, P. Hillsamer, and C. H. Kim Human Th17 Cells Share Major Trafficking Receptors with Both Polarized Effector T Cells and FOXP3+ Regulatory T Cells J. Immunol., January 1, 2008; 180(1): 122 - 129. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Moisan, R. Grenningloh, E. Bettelli, M. Oukka, and I-C. Ho Ets-1 is a negative regulator of Th17 differentiation J. Exp. Med., November 26, 2007; 204(12): 2825 - 2835. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Yamaguchi, K. Fujio, H. Shoda, A. Okamoto, N. H. Tsuno, K. Takahashi, and K. Yamamoto IL-17B and IL-17C Are Associated with TNF-{alpha} Production and Contribute to the Exacerbation of Inflammatory Arthritis J. Immunol., November 15, 2007; 179(10): 7128 - 7136. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Peng, G. Han, H. Shao, Y. Wang, H. J. Kaplan, and D. Sun Characterization of IL-17+ Interphotoreceptor Retinoid-Binding Protein-Specific T Cells in Experimental Autoimmune Uveitis Invest. Ophthalmol. Vis. Sci., September 1, 2007; 48(9): 4153 - 4161. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Niki, H. Takaishi, J. Takito, T. Miyamoto, N. Kosaki, H. Matsumoto, Y. Toyama, and N. Tada Administration of Cyclooxygenase-2 Inhibitor Reduces Joint Inflammation but Exacerbates Osteopenia in IL-1{alpha} Transgenic Mice Due to GM-CSF Overproduction J. Immunol., July 1, 2007; 179(1): 639 - 646. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Coppieters, P. Dewint, K. Van Beneden, P. Jacques, S. Seeuws, G. Verbruggen, D. Deforce, and D. Elewaut NKT cells: manipulable managers of joint inflammation Rheumatology, April 1, 2007; 46(4): 565 - 571. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M I Koenders, L A B Joosten, and W B van den Berg Potential new targets in arthritis therapy: interleukin (IL)-17 and its relation to tumour necrosis factor and IL-1 in experimental arthritis Ann Rheum Dis, November 1, 2006; 65(suppl_3): iii29 - iii33. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Tan, W. Huang, Q. Zhong, and P. Schwarzenberger IL-17 Receptor Knockout Mice Have Enhanced Myelotoxicity and Impaired Hemopoietic Recovery Following Gamma Irradiation J. Immunol., May 15, 2006; 176(10): 6186 - 6193. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. I. Koenders, E. Lubberts, F. A. J. van de Loo, B. Oppers-Walgreen, L. van den Bersselaar, M. M. Helsen, J. K. Kolls, F. E. Di Padova, L. A. B. Joosten, and W. B. van den Berg Interleukin-17 Acts Independently of TNF-{alpha} under Arthritic Conditions J. Immunol., May 15, 2006; 176(10): 6262 - 6269. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Kramer, L. Yi, F. Shen, A. Maitra, X. Jiao, T. Jin, and S. L. Gaffen Cutting Edge: Evidence for Ligand-Independent Multimerization of the IL-17 Receptor J. Immunol., January 15, 2006; 176(2): 711 - 715. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. C. Jenmalm, H. Cherwinski, E. P. Bowman, J. H. Phillips, and J. D. Sedgwick Regulation of Myeloid Cell Function through the CD200 Receptor J. Immunol., January 1, 2006; 176(1): 191 - 199. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Lubberts, P. Schwarzenberger, W. Huang, J. R. Schurr, J. J. Peschon, W. B. van den Berg, and J. K. Kolls Requirement of IL-17 Receptor Signaling in Radiation-Resistant Cells in the Joint for Full Progression of Destructive Synovitis J. Immunol., September 1, 2005; 175(5): 3360 - 3368. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. I. Koenders, E. Lubberts, B. Oppers-Walgreen, L. van den Bersselaar, M. M. Helsen, F. E. Di Padova, A. M.H. Boots, H. Gram, L. A.B. Joosten, and W. B. van den Berg Blocking of Interleukin-17 during Reactivation of Experimental Arthritis Prevents Joint Inflammation and Bone Erosion by Decreasing RANKL and Interleukin-1 Am. J. Pathol., July 1, 2005; 167(1): 141 - 149. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A. Latham, K. B. Whittington, R. Zhou, Z. Qian, and E. F. Rosloniec Ex Vivo Characterization of the Autoimmune T Cell Response in the HLA-DR1 Mouse Model of Collagen-Induced Arthritis Reveals Long-Term Activation of Type II Collagen-Specific Cells and Their Presence in Arthritic Joints J. Immunol., April 1, 2005; 174(7): 3978 - 3985. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. T. Nardelli, M. A. Burchill, D. M. England, J. Torrealba, S. M. Callister, and R. F. Schell Association of CD4+ CD25+ T Cells with Prevention of Severe Destructive Arthritis in Borrelia burgdorferi-Vaccinated and Challenged Gamma Interferon-Deficient Mice Treated with Anti-Interleukin-17 Antibody Clin. Vaccine Immunol., November 1, 2004; 11(6): 1075 - 1084. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Nakae, A. Nambu, K. Sudo, and Y. Iwakura Suppression of Immune Induction of Collagen-Induced Arthritis in IL-17-Deficient Mice J. Immunol., December 1, 2003; 171(11): 6173 - 6177. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Burchill, D. T. Nardelli, D. M. England, D. J. DeCoster, J. A. Christopherson, S. M. Callister, and R. F. Schell Inhibition of Interleukin-17 Prevents the Development of Arthritis in Vaccinated Mice Challenged with Borrelia burgdorferi Infect. Immun., June 1, 2003; 71(6): 3437 - 3442. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Nakae, S. Saijo, R. Horai, K. Sudo, S. Mori, and Y. Iwakura IL-17 production from activated T cells is required for the spontaneous development of destructive arthritis in mice deficient in IL-1 receptor antagonist PNAS, May 13, 2003; 100(10): 5986 - 5990. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. D. Silverman, D. O. Zamora, Y. Pan, P. V. Texeira, S.-H. Baek, S. R. Planck, and J. T. Rosenbaum Constitutive and Inflammatory Mediator-Regulated Fractalkine Expression in Human Ocular Tissues and Cultured Cells Invest. Ophthalmol. Vis. Sci., April 1, 2003; 44(4): 1608 - 1615. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Lubberts, L. van den Bersselaar, B. Oppers-Walgreen, P. Schwarzenberger, C. J. J. Coenen-de Roo, J. K. Kolls, L. A. B. Joosten, and W. B. van den Berg IL-17 Promotes Bone Erosion in Murine Collagen-Induced Arthritis Through Loss of the Receptor Activator of NF-{kappa}B Ligand/Osteoprotegerin Balance J. Immunol., March 1, 2003; 170(5): 2655 - 2662. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Ferretti, O. Bonneau, G. R. Dubois, C. E. Jones, and A. Trifilieff IL-17, Produced by Lymphocytes and Neutrophils, Is Necessary for Lipopolysaccharide-Induced Airway Neutrophilia: IL-15 as a Possible Trigger J. Immunol., February 15, 2003; 170(4): 2106 - 2112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. A. Gracie, S. E. Robertson, and I. B. McInnes Interleukin-18 J. Leukoc. Biol., February 1, 2003; 73(2): 213 - 224. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. K. Kolls, S. T. Kanaly, and A. J. Ramsay Interleukin-17: An Emerging Role in Lung Inflammation Am. J. Respir. Cell Mol. Biol., January 1, 2003; 28(1): 9 - 11. [Full Text] [PDF]
|
|
|
|
|
|
J. Maertzdorf, A. D. M. E. Osterhaus, and G. M. G. M. Verjans IL-17 Expression in Human Herpetic Stromal Keratitis: Modulatory Effects on Chemokine Production by Corneal Fibroblasts J. Immunol., November 15, 2002; 169(10): 5897 - 5903. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P J Koshy, N Henderson, C Logan, P F Life, T E Cawston, and A D Rowan Interleukin 17 induces cartilage collagen breakdown: novel synergistic effects in combination with proinflammatory cytokines Ann Rheum Dis, August 1, 2002; 61(8): 704 - 713. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
