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Tenosynovitis and osteoclast formation as the initial preclinical changes in a murine model of inflammatory arthritis.

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ARTHRITIS & RHEUMATISM
Vol. 56, No. 1, January 2007, pp 79–88
DOI 10.1002/art.22313
© 2007, American College of Rheumatology
Tenosynovitis and Osteoclast Formation as the
Initial Preclinical Changes in a Murine Model of
Inflammatory Arthritis
Silvia Hayer,1 Kurt Redlich,1 Adelheid Korb,1 Sonja Hermann,2 Josef Smolen,1
and Georg Schett3
Objective. To determine the nature of the initial
changes of joint inflammation occurring before, at the
time of, and shortly after onset of clinically apparent
arthritis.
Methods. Human tumor necrosis factor (TNF)–
transgenic mice were assessed for clinical, histologic,
immunophenotypic, serologic, and molecular changes at
the preclinical phase of arthritis, at the onset of disease,
and at the stage of early clinical disease. In addition, the
effects of a genetic osteoclast deficiency and pharmacologic inhibition of TNF were studied in these initial
phases of disease.
Results. Initial articular changes were observed
even before the start of clinical symptoms. Infiltration of
the tendon sheaths by granulocytes and macrophages as
well as formation of osteoclasts next to the inflamed
tendon sheaths were the first pathologic events. Tenosynovitis rapidly led to remodeling of the sheaths into
pannus-like tissue, which formed osteoclasts that invaded the adjacent mineralized cartilage. Early lesions
were associated with up-regulation of interleukin-1
(IL-1) and IL-6 as well as activation of p38 MAPK and
ERK. In contrast, absence of osteoclasts led to uncoupling of tenosynovitis from invasion into cartilage and
bone. TNF blockade also attenuated the pathologic
changes associated with tenosynovitis.
Conclusion. Structural damage begins even before the onset of clinical symptoms of arthritis and
involves the tendon sheaths as well as adjacent cartilage
and bone. These results suggest that tenosynovitis is an
initiating feature of arthritis and that joint destruction
starts right from the onset of disease. Our findings thus
underscore the importance of immediate initiation of an
effective therapy in patients with rheumatoid arthritis.
Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by synovial hyperplasia,
cartilage damage, and bone erosions, leading to progressive disability. Information on the character of the initial
destructive events in arthritis is limited, since the affected structures are not directly accessible in patients
with very early disease, and imaging techniques such as
radiography and magnetic resonance imaging (MRI) do
not provide insight into the cellular processes involved in
the initial destructive process. Thus, it is unclear whether
joint destruction accompanies inflammation of the joints
right from disease onset or whether there is a certain lag
period between onset of inflammation and structural
damage. The latter has been supported by evidence of
synovial inflammation occurring even in the absence of
clinically apparent symptoms of arthritis (1).
Chronic arthritic diseases such as RA usually lead
to joint damage after a short period of time. Even
comparatively insensitive techniques such as plain radiography show destructive changes within 6 months from
disease onset in more than 50% of patients with RA (2).
The destructive processes in RA depend on a complex
interplay of synovial fibroblasts, lymphocytes, macrophages, and monocyte-derived osteoclasts (3,4). The
documented invasive properties of synovial fibroblasts
Dr. Schett’s work was supported by a START Program award
from the Austrian Science Fund.
1
Silvia Hayer, PhD, Kurt Redlich, MD, Adelheid Korb, MD,
Josef Smolen, MD: Medical University of Vienna, Vienna, Austria;
2
Sonja Hermann, MD: University of Erlangen-Nuremberg, Erlangen,
Germany; 3Georg Schett, MD: Medical University of Vienna, Vienna,
Austria, and University of Erlangen-Nuremberg, Erlangen, Germany.
Address correspondence and reprint requests to Georg
Schett, MD, Department of Internal Medicine III and Institute for
Clinical Immunology, University of Erlangen-Nuremberg, Krankenhausstrasse 12, D-91054 Erlangen, Germany. E-mail: georg.schett@
med3.imed.uni-erlangen.de.
Submitted for publication June 28, 2006; accepted in revised
form September 29, 2006.
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HAYER ET AL
include production of tissue-degrading enzymes such as
cathepsins and matrix metalloproteinases (5). In addition, macrophages, activated synovial fibroblasts, and
lymphocytes contribute to this process by synthesizing
proinflammatory cytokines and chemokines. Tumor necrosis factor (TNF) is a key cytokine in RA (6), and
selective overexpression of TNF in mice is sufficient for
the development of destructive arthritis (7). Of note,
synovial fibroblasts and lymphocytes drive the differentiation of osteoclasts from monocyte precursor cells via
the expression of RANKL (8–11). Osteoclasts are cells
that are specifically designed to resorb bone (12).
The purpose of this study was to investigate the
nature and timing of joint destruction in chronic arthritis. We hypothesized that microscopic signs of joint
destruction are present as early as the onset of clinically
apparent disease. To address this question, we used
human TNF-transgenic (HuTNF-Tg) mice as an experimental arthritis model. This model is characterized by
1) a chronic progressive course of disease, 2) a symmetric polyarthritis with predominant involvement of
the small joints, and 3) a destructive phenotype, all of
which are features that closely resemble those of human
RA. Structural changes in the joints were assessed at the
stage of preclinical disease, at clinical onset of disease,
and during early disease. Alterations in the periarticular
tissue, such as the tendons, as well as in the articular
cartilage and bone were assessed during these 3 initial
phases of arthritis. Our results show that tenosynovitis is
the first pathologic event manifest in arthritis. Moreover, tenosynovitis precipitates the rapid destruction of
the adjacent juxtaarticular bone even in these very early
stages of disease.
MATERIALS AND METHODS
Animals. Heterozygous HuTNF-Tg mice (Tg197
strain, C57BL/6 genetic background) spontaneously develop
inflammatory, destructive arthritis upon constitutive HuTNF
overexpression. Both the genotype and phenotype of these
mice have been described previously in more detail (7). Briefly,
the animals develop joint swelling in the small joints of the
front and hind paws between weeks 5 and 6 after birth. This
chronic polyarthritis continuously progresses over time and
finally results in severe joint damage. In this experiment, young
inbred littermates were assessed weekly for the onset of clinical
symptoms of arthritis starting 3 weeks after birth.
Based on the onset of paw swelling and loss of grip
strength, early arthritis in 3–6-week-old HuTNF-Tg mice was
categorized in 1 of the 3 stages, as follows: 1) the preclinical
stage, with no signs of joint swelling or loss of grip strength,
2) the disease onset stage, characterized by the appearance
of the first clinical signs of arthritis, and 3) the early arthritis
stage, characterized by signs and symptoms of the disease
1 week after disease onset. Each of the groups comprised
6 animals. Moreover, 6 additional HuTNF-Tg mice were
treated with anti-TNF antibodies (infliximab; Centocor, Leiden, The Netherlands) at 10 mg/kg 3 times per week from
week 4 to week 6. Six c-fos⫺/⫺ ⫻ HuTNF-Tg mice, which are
completely deficient in osteoclast formation, were also studied.
(Generation of c-fos ⫺/⫺ ⫻ HuTNF-Tg mice has
been described previously [13].) All animals were killed by
cervical dislocation and the blood was withdrawn by heart
puncture. Animal procedures were approved by the local ethics
committee.
Clinical assessment. Clinical signs of arthritis, including grip strength and paw swelling, were assessed weekly in the
mice starting 3 weeks after birth. Paw swelling was assessed by
using a well-established semiquantitative score: 0 ⫽ no swelling, 1 ⫽ mild swelling of the toes and ankle, 2 ⫽ moderate
swelling of the toes and ankle, and 3 ⫽ severe swelling of the
toes and ankle. Grip strength was also recorded using a
semiquantitative score: 0 ⫽ normal grip strength, ⫺1 ⫽ mildly
reduced grip strength, ⫺2 ⫽ severely reduced grip strength,
and ⫺3 ⫽ no grip at all.
Conventional histology and immunohistochemistry.
Decalcified paraffin-embedded 2-␮m sections of the paws
were stained with hematoxylin and eosin for evaluation of
inflammation, with toluidine blue for cartilage proteoglycan
loss, and with tartrate-resistant acid phosphatase (TRAP)
(leukocyte staining kit; Sigma, St. Louis, MO) for the amount
of osteoclasts and severity of bone erosion. To investigate cell
populations, immune phenotyping was carried out on T cells
(anti-CD3, 1:50 dilution; Novo Castra Laboratories, Newcastle, UK), B cells (anti-CD45 receptor, 1:200 dilution; BD
Biosciences PharMingen, San Diego, CA), neutrophils (clone
7/4, 1:2,000 dilution; Serotec, Oxford, UK), and macrophages
(clone F4/80, 1:300 dilution; Serotec). For further immunohistochemical analyses, sections were stained with interleukin-1
(IL-1) antibodies (1:25 dilution; R&D Systems, Minneapolis,
MN) and IL-6 antibodies (1:25 dilution; Acris Antibodies,
Hiddenhausen, Germany). Sections were pretreated with proteinase K (0.5 ␮g/ml for 5 minutes at 37°C; Roche Diagnostics,
Mannheim, Germany) for granulocytes, macrophages, T cells,
and IL-6 staining, or were pretreated with heat (for 20 minutes
at 96°C) for IL-1 staining, or were left untreated for B cell
staining. Blocking of endogenous peroxidase was done with
0.3–3% hydrogen peroxide in phosphate buffered saline for
10 minutes.
Sections were then incubated for 30 minutes with
biotinylated species-specific secondary antibodies as follows:
rabbit anti-rat antibody for the detection of granulocytes,
macrophages, B lymphocytes, and T lymphocytes, goat antirabbit antibody for the detection of IL-6, and rabbit anti-goat
antibody for the detection of IL-1 (all from Vector Laboratories, Burlingame, CA). Subsequently, sections were incubated
for 10 minutes with StreptABComplex/horseradish peroxidase (HRP) (Dako, Glostrup, Denmark) for the detection of
macrophages and B lymphocyte, T lymphocyte, IL-1, and IL-6
staining, or with avidin–biotin–HRP complex (Vectastain
ABC kit; Vector Laboratories) for the detection of granulocytes. Antigen-expressing cells were visualized by incubation
with 3,3-diaminobenzidine (Sigma), resulting in brown staining. Areas of synovial inflammation, bone erosion, cartilage
damage, and bone marrow infiltrates were quantified by
PRECLINICAL TENOSYNOVITIS AND OSTEOCLAST FORMATION
81
Figure 1. Definition of different phases of very early inflammatory arthritis and characterization of inflammation. A and B,
Based on the severity of paw swelling (A) and reduction in grip strength (B), 3 phases of very early arthritis were defined: 1)
the preclinical stage, before the onset of clinical symptoms of arthritis (weeks 3–4), 2) the clinical onset of arthritis, with start
of swelling and deterioration of grip strength (weeks 4–5), and 3) early arthritis, up to 1 week after disease onset (weeks 5–6).
Results are the mean and SEM in human tumor necrosis factor–transgenic (HuTNF-Tg) mice versus wild-type (WT) mice. C
and D, Hematoxylin and eosin–stained paw sections were assessed for signs of inflammation in wild-type as well as HuTNF-Tg
mice in various different phases of early disease, characterized by pannus formation in the hind paws at the onset of
inflammation (C) and tenosynovitis of the long peroneal muscle (D). Boxes in C are shown at higher magnification in D.
(Original magnification ⫻ 25 in C; ⫻ 100 in D.)
histomorphometry using an Axioskop 2 microscope (Zeiss,
Oberkochen, Germany) and OsteoMeasure Analysis software
(OsteoMetrics, Decatur, GA).
Analysis of kinase activity by Western blotting. Protein
extracts were prepared from the hind paws of 9 HuTNF-Tg
mice (3 from each time point) and 3 wild-type mice by mincing
the tissue in lysis buffer containing 20 mM HEPES, pH 7.9,
0.4M NaCl, 1.5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA,
0.1 mM EGTA, 20% glycerol, and proteinase and phosphatase
inhibitors (both from Sigma) with an Ultra-Thurrax homogenizer. The extracts were centrifuged for 15 minutes at 14,000
revolutions per minute, separated by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis, and transferred onto nitrocellulose membranes. After blocking, membranes were stained
with polyclonal antibodies against the phosphorylated forms
of p38 MAPK, ERK (p44/42 MAPK), and SAPK/JNK (all
from Cell Signaling, Beverly, MA). For control purposes,
antibodies against total p38 MAPK, ERK, SAPK/JNK (Cell
Signaling), and actin (Sigma) were used. Detection was performed using HRP-conjugated secondary antibodies (Dako,
Copenhagen, Denmark) and an enhanced chemiluminescence
detection kit (ECL Western Blotting Detection Reagents;
Amersham Biosciences, Buckinghamshire, UK).
Enzyme-linked immunosorbent assay (ELISA). Serum
levels of IL-1 and IL-6 in the 3–6-week-old mice were analyzed
by quantikine ELISA (R&D Systems) in accordance with the
manufacturer’s protocol.
Statistical analysis. Results are expressed as the
mean ⫾ SEM. Group mean values were compared by MannWhitney U test. P values less than or equal to 0.05 were
considered significant.
RESULTS
Differentiation into the preclinical, disease onset, and early arthritis stages. Three different phases of
very early arthritis based on a weekly assessment of paw
swelling and grip strength were defined in HuTNF-Tg
mice (Figures 1A and B). The preclinical stage, defined
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HAYER ET AL
Figure 2. Immune phenotyping, cytokine expression, and intracellular signaling in the initial phases of tumor
necrosis factor–mediated arthritis. Cellular distribution of the inflamed tendon sheaths was determined by
immunohistochemical staining for granulocytes (A), macrophages (B), T lymphocytes (C), and B lymphocytes (D)
in human tumor necrosis factor–transgenic mice in the various phases of early disease. Brown cells indicate
positive labeling. (Original magnification ⫻ 100.)
by the absence of joint swelling and by normal (score 0)
grip strength, was generally found between weeks 3 and
4 after birth. The disease onset stage, defined by the
appearance of the first signs of joint swelling and start of
deterioration of grip strength, could usually be observed
between weeks 4 and 5 after birth. The third stage, early
arthritis, was characterized by continuing mild clinical
signs and symptoms 1 week thereafter (at weeks 5–6
after birth).
Manifestation of the first inflammatory event,
tenosynovitis, at the preclinical stage. To address the
first inflammatory musculoskeletal changes occurring
upon TNF overexpression, we analyzed the paw sections
of HuTNF-Tg mice during the 3 disease stages (Figures
1C and D). Interestingly, in preclinical disease, the
initial pathologic change was a tendon effusion rather
than synovitis (Figure 1C). Shortly thereafter, in the
disease onset stage, a multitude of inflammatory cells
PRECLINICAL TENOSYNOVITIS AND OSTEOCLAST FORMATION
ingressed into the tendon sheath, culminating, by the
early disease stage, in a massive hyperplasia of the
synovialis of the tendon sheath (Figure 1D). The thickness of the tendon sheath, in particular of the long
peroneal muscle of the hind paw, was markedly increased in HuTNF-Tg mice compared with wild-type
mice from 3 weeks onward.
Formation of inflammatory pannus at disease
onset. In conjunction with increased cellular influx into
the tendon sheaths at disease onset, progressive hyperplasia of the joint synovial lining was observed, whereas
no increased thickness of the joint synovial lining was
seen during preclinical disease. Inflammatory pannus
formation occurred simultaneously with cell influx and
synovial hyperplasia of the tendon sheaths and dominated at sites adjacent to the initially inflamed tendon
sheaths. Inflammatory pannus formation further increased significantly in early disease compared with
that at disease onset. (For immunohistochemical images
and quantitative data, a printout is available from the
corresponding author upon request.)
Influx of granulocytes and macrophages prior to
development of tenosynovitis. We next assessed the cell
populations participating in the initial tendon sheath
inflammation. Interestingly, the dominant cell population causing tenosynovitis was granulocytes (Figure 2A).
Single granulocytes were found in the synovium of the
tendon sheath, but most of them accumulated in the
effusion, resulting in a dramatic increase in cell numbers. Apart from granulocytes, macrophages were also
found in the effusion, accumulating in early clinical
disease (Figure 2B). In contrast, only a few T lymphocytes were present in the effusions (Figure 2C). B cells
were absent in initial tenosynovitis and did not accumulate (Figure 2D). (For quantitative data on the different
leukocyte populations invading the tendon sheath, a
printout is available from the corresponding author
upon request.)
Expression of IL-1 and IL-6 in early inflammatory tenosynovitis. To further characterize the initial
events of inflammatory arthritis occurring in the
HuTNF-Tg mice, we investigated the expression of 2
major TNF-dependent inflammatory cytokines, IL-1 and
IL-6. Interestingly, even the first effusing cells in initial
(preclinical) tenosynovitis expressed IL-1, suggesting
that IL-1 is not only induced very early in disease but
also linked to the initial process, since other areas of the
joint did not express IL-1 at this stage (Figure 3A).
IL-1–positive cells were mainly neutrophils in the synovial tendon sheaths, whereas the mesenchymal lining of
these sheaths was IL-1 negative. Later in the early stage,
83
Figure 3. Cytokine expression in the initial phases of tumor necrosis
factor (TNF)–mediated arthritis. Expression of cytokines in the inflamed tendon sheaths was determined by immunohistochemical staining for interleukin-1 (IL-1) (A) and IL-6 (B), and serum levels of IL-1
(C) and IL-6 (D) were determined by enzyme-linked immunosorbent
assay in wild-type (WT) and human TNF–transgenic (HuTNF-Tg)
mice in various phases of early disease. Results are the mean and SD
numbers of positive cells. ⴱ ⫽ P ⬍ 0.05 versus HuTNF-Tg mice at
disease onset and/or preclinical stage.
IL-1–expressing cells were also located in the inflammatory pannus and at sites of bone erosion in early disease.
IL-6 was also found to be expressed by neutrophils in the
synovial effusions right from the preclinical stage of
disease (Figure 3B). In contrast to the pattern of IL-1
expression, IL-6 was also expressed by mesenchymal
cells lining the inflamed tendon sheaths. Similarly, expression of IL-6 further increased with disease progression. (For immunohistochemical images, a printout is
available from the corresponding author upon request.)
This elevation in local expression of IL-1 and
IL-6 also resulted in an elevation of the serum levels of
IL-1 and IL-6, which significantly increased at the stage
of early disease and followed the local increase of these
2 cytokines (Figures 3C and D). Thus, the rise in levels
of these cytokines in the serum paralleled the increasing
cytokine expression in the tendon sheaths.
Increased expression and activation of MAPKs
in early inflammatory arthritis. To better characterize
the molecular changes at the initiation of arthritis,
articular tissue of HuTNF-Tg mice was investigated for
the expression and activation of members of the MAPK
family. Protein expression of activated p38 MAPK was
unchanged at the start of disease, whereas activation of
p38 MAPK became evident in preclinical disease and
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HAYER ET AL
Figure 4. Onset of bone erosion and cartilage damage in tumor necrosis factor (TNF)–mediated arthritis. A and B,
Tartrate-resistant acid phosphatase–stained sections showing the formation of mononucleated osteoclast precursors (A),
multinucleated osteoclasts (purple staining indicated by arrows in B), and bone erosions (B) in human TNF–transgenic mice
in various phases of early disease as compared with wild-type mice. Boxes in A are shown at higher magnification in B (original
magnification ⫻ 25 in A; ⫻ 400 in B). C, Formation of bone marrow inflammation demonstrated by CD45 receptor–positive
B lymphocyte staining (brown color indicated by arrows) (original magnification ⫻ 400).
increased continuously over the course of early disease.
In contrast, total SAPK/JNK expression did not show
any increase at the stage of early arthritis and was not
accompanied by an increase in activation as disease
progressed. Interestingly, ERK increased right from the
preclinical stage of disease and also showed an increased
activation over time. (For Western blot images of the
kinase activities, a printout is available from the corresponding author upon request.)
Formation of primary bone erosions preceding
the onset of clinical signs of arthritis. To determine the
onset of bone erosion, we next searched for osteoclasts
emerging in the synovial tissue. Interestingly, the first
mononuclear TRAP⫹ cells appeared at the preclinical
stage of disease, at sites where the inflamed tendon
sheaths passed by the cartilage–pannus junction (Figures
4A and B). These initial preosteoclasts were associated,
even at this earliest stage, with what appeared to be
shallow erosions of the mineralized cartilage. TRAP⫹
cells then rapidly increased in number, size, and nucleation during the stages of disease onset and early
arthritis, and these changes were associated with an
increase in the size of bone erosions.
To investigate the participation of juxtaarticular
bone marrow in the onset of disease, we investigated this
compartment by immunopleiotypic labeling. Cellular
accumulations in the bone marrow formed upon disease
onset but not in the earlier, preclinical stage (Figure 4C).
The number and size of the bone marrow infiltrates
increased at the early arthritis stage. These abnormalities were almost exclusively composed of B cells. (For
quantitative data on these destructive changes, a printout is available from the corresponding author upon
request.)
Onset of cartilage damage after bone destruction
in TNF-mediated arthritis. To determine the pattern of
onset of cartilage damage, we evaluated the content of
proteoglycans at the articular surface as well as the
structural integrity of cartilage. The cartilage surface
decreased gradually during these early stages of arthritis,
but damage in the cartilage occurred much slower than
in synovial tissue and bone. Thus, even in early arthritis,
PRECLINICAL TENOSYNOVITIS AND OSTEOCLAST FORMATION
the cartilage surface, although somewhat diminished,
was not significantly reduced as compared with that in
wild-type mice. Loss of proteoglycans, however, started
early, at the stage of disease onset, and continued to
proceed to structural damage of cartilage. Nonetheless,
initiation of the pathologic changes in the cartilage
followed both the development of tenosynovitis and the
formation of osteoclasts, the latter being the initial
events in this inflammatory arthritis model. (For immunohistochemical images and quantitative data, a printout
is available from the corresponding author upon request.)
Effects of antiresorptive and antiinflammatory
therapy on early arthritis. On the basis of the early appearance of osteoclasts, we wanted to assess whether
complete lack of these cells could alter the course of
early inflammatory arthritis. In c-fos–deficient HuTNF-Tg
mice, which lack osteoclasts, we found that tenosynovitis became apparent at the onset of disease, as characterized by inflamed tendon sheaths next to the
periosteal bone surface. However, inflamed tissue was
unable to create osteoclasts and thus failed to invade the
subchondral bone.
In contrast, early initiation of TNF blockade
affected the development of tenosynovitis by reducing
cellular infiltration and edema. Moreover, formation of
synovial osteoclasts was decreased. Thus, TNF blockade
has a dual mode of action with respect to joint damage
in inflammatory arthritis. It blocks inflammatory tenosynovitis as well as osteoclast formation at the regions
prone to bone erosions. (For immunohistochemical images and quantitative data, a printout is available from
the corresponding author upon request.)
DISCUSSION
The early phases of RA have not been studied
extensively. Although it is known that the pattern of
disease onset may differ between patients, in the majority of patients with RA the disease starts insidiously (14).
Not infrequently, tenosynovitis accompanies the early
steps of RA (15–17). The sequence of the earliest
pathologic events is still unknown. It has been postulated
that an influx of inflammatory cells into the synovium
precedes the development of the signs and symptoms of
the disease, since frank synovitis could be found in the
joints of RA patients who were clinically unaffected (1).
It is also evident that immune system abnormalities
precede the development of RA, since autoantibodies
can be detected months to years before onset of the
disease (18–20). Moreover, ⬃10% of RA patients have
evidence of bone erosions on plain radiographs as early
85
as 8 weeks after disease onset (Machold K: personal
communication). These and other observations have led
to the concept of prearthritis (21).
However, the events governing the changes that
occur before arthritis, i.e., at the time that inflammatory
joint disease becomes manifest, cannot be easily studied
in humans, since at the preclinical stage, tissue is not
easily accessible and standard imaging techniques, including MRI, have an insufficient resolution to discern
changes on the cellular level. To address this question,
we therefore studied an animal model that closely
resembles human RA in terms of overexpression of TNF
and the characteristics of inflammation and bone and
cartilage destruction.
With the aim of studying the first pathologic
events of TNF-mediated inflammatory arthritis in the
joint, we used the HuTNF-Tg mouse model of arthritis
and screened for the onset of inflammation and structural damage before, at the time of, and shortly after
disease onset. In fact, even in animal models, the
knowledge regarding these initial phases of arthritis is
scarce. Both collagen-induced and adjuvant-induced arthritis have a very rapid disease onset and progress
quickly, making the dissection of these initial phases of
arthritis difficult (3,22). However, it is evident from
these models that highly active arthritis needs only a few
days to induce destruction of the affected joint (3). The
disease in HuTNF-Tg mice has a mild and rather
unspectacular onset, but it continuously progresses over
time, leading to accumulation of an increasing amount
of joint damage. Thus, with the use of this model, we
were able to better study these initial phases of disease
and relate them to the onset of clinical symptoms in
more detail.
Interestingly, our study revealed that tendons are
the earliest structures affected by inflammation (Figure
5). These structures are subject to the most prominent
shear forces and are under prolonged mechanical load.
Tendons are surrounded by synovial tissue, and the
tendon sheaths have a cell composition similar to that of
the synovial lining. Accumulation of fluid as well as
effusion of inflammatory cells, primarily granulocytes
and macrophages, into these structures is the first pathologic event induced by overexpression of TNF. Many
investigators have speculated as to why these tendon
sheaths are the first structures affected by chronic
inflammation, but mechanical factors are likely to play a
key role in this process. Ultimately, a complete remodeling of the tendon sheaths to pannus-like synovial tissue
occurs, thereby completely filling the synovial space with
inflamed tissue. This obviously leads to massive distur-
86
HAYER ET AL
Figure 5. Model of arthritis onset in mice. Arthritis starts before onset of clinical disease, with infiltration
of tendon sheaths (purple) by granulocytes and monocytes, leading to tenosynovitis. Tenosynovitis
precipitates local osteoclast formation (red), in which tendons pass by the joint edges, thus inducing
resorption of mineralized cartilage and bone. As tenosynovitis becomes more severe, the synovial
membrane of the joint also becomes inflamed and hyperplastic (green), which is reflected in the onset of
clinical disease. Structural damage further increases (light yellow). In the next step, tendon sheaths
become further infiltrated and more pronounced inflammation of the synovial membrane occurs, which
leads to an increase in clinical symptoms and acceleration of joint damage.
bance of the tendon and ultimately impedes joint function.
Moreover, the tendons use the bony ends and
their edges as anchors to increase function, thus bringing
these structures into close contact with sites predisposed
to the development of bone erosions. These areas in
which tendon sheaths, mineralized cartilage, and subchondral bone are in close contact are very distinct. In
these contact regions, the first osteoclasts appear and
start to resorb subchondral bone and mineralized cartilage (10,23). Thus, even before the onset of synovial
inflammation, osteoclasts start their destructive work
and pave the way for early B cell accumulation in the
adjacent bone marrow, which is closely linked to breaking of the cortical bone barrier (24,25). Since osteoclasts
appear even before the first clinical signs of arthritis,
destructive changes begin to occur even before arthritis
is clinically apparent in the patients. This finding further
strengthens the concept of a prearthritic state and
confirms that there is a very narrow window of opportunity for achieving full remission of RA without any
sequelae (26).
The activity of TNF leads to activation of MAPK
cascades, such as p38 MAPK and ERK, very early in the
disease. This is consistent with the well-documented
activation of MAPK families in human RA (27–30).
Thus, even in the preclinical disease phase, the joints of
the HuTNF-Tg mice showed an activation of these 2
MAPK members, suggesting that p38 MAPK and ERK
mediate the cellular effects of TNF right from the start
of molecular onset of arthritis. As a matter of fact, p38
MAPK plays a major role in facilitating the cytokine
activation induced by TNF. Induction of proinflammatory mediators such as IL-1 and IL-6 by TNF is mediated
by activation of p38 MAPK (31). Of note, these cytokines are inducibly expressed in these initial stages of
arthritis and appear to be selectively linked to the areas
of onset of inflammation, such as the cellular infiltration
of the tendon sheaths. IL-1 and IL-6 are subsequently
expressed extensively throughout the newly formed inflamed synovial tissue. We found that this increased
expression of IL-1 and IL-6 was paralleled by a rise in
the serum levels of these 2 cytokines. Early activation of
ERK, in contrast, might influence the survival of cells in
the synovial membrane. ERK activation by TNF plays an
important role in the maintenance of cell survival and
the inhibition of apoptosis (32). In fact, apoptosis is low
in the synovial membrane, suggesting that cytokines like
TNF act on intracellular survival factors rather than
apoptosis-inducing signaling molecules (33,34).
In the present study, we also observed that damage of unmineralized surface cartilage occurred subsequent to, and not before or concomitant with, tenosynovitis and osteoclast formation. In contrast to osteoclastmediated damage, which affects mineralized structures,
breakdown of the surface cartilage depends on cytokine
and enzyme production of cells in the joint cavity and
the synovial lining. Cartilage damage was linked to a
PRECLINICAL TENOSYNOVITIS AND OSTEOCLAST FORMATION
certain level of inflammation and structural organization
of the synovial surface (lining layer), suggesting that a
threshold of synovial inflammation is critical for matrix
resorption. Thus, whereas destruction of mineralized
tissue was linked to tenosynovitis and occurred very
early in the disease, cartilage damage started later and
was linked to inflammation of the synovial membrane.
In summary, we have shown that tenosynovitis is
the first structural change to occur in TNF-mediated
arthritis. Regions of close interaction between the tendons and mineralized tissue allow the spread of inflammatory changes to the joints, particularly leading to the
resorption of mineralized cartilage and bone. Importantly, these changes occur before the first clinical signs
of joint swelling are apparent. Our data suggest that
newly occurring tenosynovitis should be taken seriously,
since it might be followed by the development of RA.
Moreover, structural changes in the joints are a very
early feature of disease, starting right from its subclinical
onset. Thus, the time window to protect joints from
damage may, in fact, be very short, and this knowledge
should give impetus to the efforts for early initiation of
targeted therapy in patients with RA.
87
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ACKNOWLEDGMENTS
We thank Thomas Pangerl, Birgit Türk, and Margarete Tryniecki for excellent technical assistance, Prof. Erwin
Wagner (Research Institute for Molecular Pathology, Vienna,
Austria) for providing the c-fos–deficient mice, and Prof.
George Kollias (Alexander Fleming Biomedical Research
Center, Vari, Greece) for providing the HuTNF-Tg mice.
AUTHOR CONTRIBUTIONS
Dr. Schett had full access to all of the data in the study and
takes responsibility for the integrity of the data and the accuracy of the
data analysis.
Study design. Drs. Hayer, Redlich, Smolen, and Schett.
Acquisition of data. Drs. Hayer and Korb.
Analysis and interpretation of data. Drs. Hayer, Redlich, Hermann,
and Schett.
Manuscript preparation. Drs. Hayer, Smolen, and Schett.
Statistical analysis. Drs. Hayer and Hermann.
16.
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mode, change, initial, murine, formation, arthritis, inflammatory, tenosynovitis, preclinical, osteoclast
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