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Involvement of matrix metalloproteinases and their inhibitors in peripheral synovitis and down-regulation by tumor necrosis factor ╨Ю┬▒ blockade in spondylarthropathy.

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Vol. 50, No. 9, September 2004, pp 2942–2953
DOI 10.1002/art.20477
© 2004, American College of Rheumatology
Involvement of Matrix Metalloproteinases and Their Inhibitors
in Peripheral Synovitis and Down-Regulation by
Tumor Necrosis Factor ␣ Blockade in Spondylarthropathy
Bernard Vandooren,1 Elli Kruithof,1 David T. Y. Yu,2 Markus Rihl,3 Jieruo Gu,2
Leen De Rycke,1 Filip Van den Bosch,1 Eric M. Veys,1 Filip De Keyser,1
and Dominique Baeten1
and SpA patients. Involvement of MMPs and TIMPs in
SpA synovitis was suggested by the correlation with
cellular infiltration, vascularization, and cartilage degradation. Higher serum levels of MMPs 3 and 9 were
revealed in SpA and RA patients as compared with
healthy controls. Production of MMP-3, but not
MMP-9, in the serum reflected the presence of peripheral synovitis, as indicated by 1) the correlation
between serum levels, SF levels (which were 1,000-fold
higher than the serum levels), and synovial expression
of MMP-3, 2) the increased levels of MMP-3 in AS
patients with peripheral disease and not exclusively
axial involvement, and 3) the correlation of serum and
SF MMP-3 with parameters of synovial, but not systemic, inflammation. The modulation of the MMP/
TIMP system by tumor necrosis factor ␣ (TNF␣) blockade was confirmed by the down-regulation of all MMPs
and TIMPs in the synovium and a pronounced and
rapid decrease of serum MMP-3.
Conclusion. MMPs and TIMPs are highly expressed in SpA synovitis and mirror both the inflammatory and tissue-remodeling aspects of the local disease process. Serum MMP-3, originating from the
inflamed joint, represents a valuable biomarker for
peripheral synovitis. Modulation of the MMP/TIMP
system by infliximab could contribute to the antiinflammatory and tissue-remodeling effects of TNF␣ blockade
in SpA.
Objective. To investigate the role of matrix metalloproteinases (MMPs) and tissue inhibitors of matrix
metalloproteinases (TIMPs) in spondylarthropathy
(SpA) synovitis.
Methods. Paired samples of synovial biopsy tissue
as well as serum and synovial fluid (SF) from 41
patients with SpA and 20 patients with rheumatoid
arthritis (RA) and serum samples from 20 healthy
controls were analyzed by immunohistochemistry and
enzyme-linked immunosorbent assay for the presence of
MMPs 1, 2, 3, and 9 and TIMPs 1 and 2. In addition,
sera from 16 patients with ankylosing spondylitis (AS)
and peripheral synovitis and 17 patients with AS and
exclusively axial involvement were analyzed. An additional cohort of SpA patients was analyzed at baseline
and after 12 weeks of infliximab treatment.
Results. Staining for MMPs and TIMPs showed a
cellular and interstitial pattern in the synovial lining
and sublining layers that was similar between the RA
Dr. Rihl’s work was supported by the Deutsche Forschungsgemeinschaft (RI 119/1-1) and the Rheumatology Competence Network. Dr. De Rycke’s work was supported by the Institute for the
Promotion of Innovation by Science and Technology in Flanders
(IWT/SB/11127). Dr. Baeten’s work was supported by the Fund for
Scientific Research–Vlaanderen (FWO-Vlaanderen).
Bernard Vandooren, MD, Elli Kruithof, MD, Leen De
Rycke, MD, Filip Van den Bosch, MD, PhD, Eric M. Veys, MD, PhD,
Filip De Keyser, MD, PhD, Dominique Baeten, MD, PhD: Ghent
University Hospital, Ghent, Belgium; 2David T. Y. Yu, MD, PhD,
Jieruo Gu, MD: University of California at Los Angeles; 3Markus
Rihl, MD: Hannover Medical School, Hannover, Germany.
Drs. Vandooren and Kruithof contributed equally to this
Address correspondence and reprint requests to Elli Kruithof,
MD, Department of Rheumatology, 0K12IB, Ghent University Hospital, De Pintelaan 185, 9000 Ghent, Belgium. E-mail: [email protected]
Submitted for publication December 6, 2003; accepted in
revised form May 20, 2004.
Inflammation and structural damage of the joint
are 2 major hallmarks of autoimmune arthritides such as
rheumatoid arthritis (RA) and spondylarthropathy
(SpA). In RA, several pivotal mediators involved in
disease mechanisms have been identified, including
interleukin-1 (IL-1) and tumor necrosis factor ␣ (TNF␣)
as proinflammatory mediators (1), along with RANK
ligand (2) and matrix metalloproteinases (MMPs) as
central mediators of joint destruction. MMPs participate
in extracellular matrix (ECM) degradation by cleavage
of ECM constituents such as collagens and proteoglycans. The MMPs exert physiologic (e.g., wound healing,
embryogenesis) and pathologic (e.g., cancer, atherosclerosis) functions that are dependent on activation by
precursor zymogens and inhibition by binding to specific
inhibitors (␣2-macroglobulin, tissue inhibitors of matrix
metalloproteinases [TIMPs]). To date, more than 20
MMPs and 4 TIMPs have been recognized (3).
Expression of the MMPs and TIMPs has been
studied extensively in the synovial tissue (4–7), synovial
fluid (SF) (8–10), and serum (11–13) of patients with
RA. In the synovial compartment, they are produced by
macrophages, synovial fibroblasts, endothelial cells, neutrophils, and chondrocytes (14). Active up-regulation of
MMPs is observed after stimulation with proinflammatory cytokines such as IL-1␤ (15) and TNF␣ (15–18).
Their biologic relevance has been linked to joint destruction (19,20), angiogenesis (21), and cell trafficking (22).
MMP-1 (collagenase 1) cleaves, inter alia, type II collagen, leading to irreversible cartilage destruction,
whereas MMP-3 (stromelysin 1) cleaves proteoglycans,
fibronectin, and the smaller collagens and also activates
proMMP-1 (3). Accordingly, MMPs 1 and 3 are considered to play a pivotal role in joint destruction (14).
MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are
mediators of joint destruction (23), but additionally have
a particular role in angiogenesis (21). Moreover, in the
joint, the function of MMPs is regulated by the presence
of specific inhibitors such as TIMPs 1 and 2.
Whereas these molecular pathways have been
well analyzed in RA, the mechanisms of joint inflammation and destruction are poorly understood in SpA.
Diffuse cartilage destruction is found in both diseases.
However, focal bone erosions are far more common in
RA than in SpA, with the exception of psoriatic arthritis
(PsA) which might also manifest marked focal bone
destruction (24). In contrast, hypervascularity is a hallmark of SpA rather than RA (25,26).
Using microarray as a screening strategy for the
identification of important mediators in SpA synovitis
(27), we found a profound down-regulation of MMP-3 in
SpA synovium after infliximab treatment (data not
shown). Based on these initial results, we analyzed the
involvement of MMPs and TIMPs in SpA synovitis by
addressing the following questions. 1) What is the
baseline expression of MMPs and TIMPs in the inflamed peripheral joint? 2) Does the expression of these
mediators correlate with parameters of inflammation
and/or tissue remodeling (synovial vascularization, matrix degradation)? 3) Are serum MMP levels a good
reflection of the MMP/TIMP activity in the joint? 4)
Can the MMP/TIMP system be modulated by therapies
such as TNF␣ blockade?
Patients. To study the synovial expression of MMPs
and TIMPs in SpA, we obtained paired synovium, SF, and
serum samples from 41 patients with SpA fulfilling the European Spondylarthropathy Study Group (ESSG) criteria (28).
All patients had peripheral synovitis with involvement of at
least 1 knee joint and underwent a needle arthroscopy for
biopsy sampling. This cohort comprised patients with PsA
(defined as SpA with skin psoriasis) (n ⫽ 19), patients with
ankylosing spondylitis (AS) fulfilling the New York criteria for
AS (29) (n ⫽ 10), and patients with undifferentiated SpA
(USpA) (n ⫽ 12). As control groups, we obtained paired
synovium, SF, and serum samples from 20 patients with RA
fulfilling the American College of Rheumatology (formerly,
the American Rheumatism Association) criteria (30), as well
as serum from 20 healthy controls (10 men and 10 women). To
further analyze the relationship between MMP/TIMP expression and peripheral joint disease in SpA, we additionally
obtained serum samples from 33 patients with AS fulfilling the
New York criteria (29), of whom 17 had no peripheral joint
involvement but exhibited exclusively axial symptoms, whereas
the other 16 had at least 1 swollen joint. Table 1 summarizes
the demographic and clinical characteristics of the different
patient groups.
Finally, to analyze the effect of TNF␣ blockade on the
MMP/TIMP system, we obtained serum samples from 12
infliximab-treated and 10 placebo-treated patients with SpA
and peripheral joint involvement, at different time points
between baseline and week 12 after initiation of treatment
(31). Furthermore, in 9 of the infliximab-treated patients with
peripheral synovitis, synovial tissue samples were obtained at
baseline and week 12, as described before (32). The demographics and clinical characteristics of this cohort at baseline
and week 12 are given in Table 1.
All patients provided their written informed consent
prior to inclusion in the study. The study was approved by the
ethics committee of the local faculty of medicine.
Immunohistochemistry of synovial biopsy tissue. Synovial tissue biopsy samples (16 from each individual patient)
were obtained by needle arthroscopy of the knee as described
previously (32). Eight of the biopsy samples from each patient
were stored in formaldehyde and embedded in paraffin, and
the other 8 were snap frozen and mounted in Jung tissuefreezing medium (Leica Instruments, Nussloch, Germany) and
utilized for immunohistochemistry.
Paraffin-embedded biopsy tissues were stained with
hematoxylin and eosin for histologic analysis, which involved
determination of the mean thickness of the synovial lining
layer, vascularity of the sublining layer, cellular infiltration of
the sublining layer, and presence of lymphoid aggregates,
plasma cells, and polymorphonuclear cells. Frozen sections of
58.3 ⫾ 16.8
6.4 ⫾ 7.8
9.0 ⫾ 7.7
5.3 ⫾ 4.5
42 ⫾ 27
42.8 ⫾ 9.8
6.5 ⫾ 7.9
3.2 ⫾ 3.8
3.4 ⫾ 4.2
26 ⫾ 26
2.9 ⫾ 1.9
28 ⫾ 13
43.7 ⫾ 8.3
17.5 ⫾ 8.6
4.6 ⫾ 4.9
4.9 ⫾ 4.1
43 ⫾ 33
48.1 ⫾ 14.3
16.7 ⫾ 14.0
AS with peripheral
AS without peripheral
joint disease
joint disease (n ⫽ 17)
(n ⫽ 16)
7.3 ⫾ 5.3
2.5 ⫾ 2.2
20 ⫾ 16
1.1 ⫾ 1.2
1.2 ⫾ 2.4
6.6 ⫾ 5.2
2.3 ⫾ 2.4
24 ⫾ 30
10.9 ⫾ 11.0
1.8 ⫾ 2.2
23 ⫾ 29
49.1 ⫾ 11.0
51.0 ⫾ 10.9
8.2 ⫾ 7.1
11.2 ⫾ 10.1
59.8 ⫾ 20.2 16.8 ⫾ 13.1 64.7 ⫾ 22.9 68.1 ⫾ 27.9
Week 12
Week 12
Placebo-treated SpA
(n ⫽ 10)
Infliximab-treated SpA
(n ⫽ 12)
* Except where indicated otherwise, values are the mean ⫾ SD. SpA ⫽ spondylarthropathy; RA ⫽ rheumatoid arthritis; AS ⫽ ankylosing spondylitis; PsA ⫽ psoriatic arthritis;
USpA ⫽ undifferentiated SpA; NA ⫽ not applicable; VAS ⫽ visual analog scale; CRP ⫽ C-reactive protein; ESR ⫽ erythrocyte sedimentation rate; NSAID ⫽ nonsteroidal
antiinflammatory drug; DMARD ⫽ disease-modifying antirheumatic drug.
(n ⫽ 20)
SpA with peripheral
joint disease
(n ⫽ 41)
Clinical and descriptive features of the patients*
Age, years
Sex, no. female/male
Subtype, no. with PsA/AS/USpA
Disease duration, years
Patient’s global disease activity
assessment, mm VAS
Number of swollen joints
Serum CRP, mg/dl
ESR, mm/hour
Medication, no. taking/not taking
Table 1.
the synovial biopsy tissue were stained and scored as described
previously (25,33,34). The following mouse monoclonal antibodies (mAb) were used: anti–MMP-1 mAb (5 ␮g/ml) (collagenase, IgG2a, clone 41-1E5; Oncogene, Cambridge, MA),
anti–MMP-2 mAb (4 ␮g/ml) (gelatinase A, IgG1, clone 757F7; Oncogene), anti–MMP-3 mAb (0.5 ␮g/ml) (stromelysin 1,
IgG1, clone SL-1 IIIC4; Oncogene), anti–MMP-9 mAb (5
␮g/ml) (gelatinase B, type IV collagenase, IgG1, clone
36020.111; R&D Systems, Abingdon, UK), anti–TIMP-1 mAb
(2 ␮g/ml) (IgG1, clone 102D1; Oncogene), and anti–TIMP-2
mAb (2 ␮g/ml) (IgG1␬, clone T2-101; Oncogene). After
incubation with the primary antibody, sections were sequentially incubated with a biotinylated second antibody, with a
streptavidin–horseradish peroxidase link, and finally with
amino-ethyl-carbazole substrate as chromogen. Parallel sections were incubated with irrelevant isotype and
concentration-matched mAb as negative controls.
Sections were coded and analyzed semiquantitatively
on a 4-point scale (ranging 0–3) by 2 independent observers
(BV and EK) who were blinded to the diagnosis and clinical
data (25,33,34). Global interobserver agreement on the different markers was 80% for the lining layer (mean correlation
coefficient 0.675, P ⬍ 0.001) and 70% for the sublining layer
(mean correlation coefficient 0.760, P ⬍ 0.001). Of particular
interest in this study was MMP-3, and the interobserver
agreement on MMP-3 expression in the lining and sublining
layers was a mean 0.718 (P ⬍ 0.001) and 0.830 (P ⬍ 0.001),
respectively. Moreover, MMP-3 samples were scored twice by
the same observer, resulting in a global intraobserver mean
correlation coefficient of 0.672 (P ⬍ 0.001) for the lining layer
and 0.686 (P ⬍ 0.001) for the sublining layer.
Immunoassays. Serum and SF levels of MMP-1,
MMP-2, MMP-3, MMP-9, TIMP-1, and TIMP-2 were measured by enzyme-linked immunosorbent assay (ELISA) (Biotrak; Amersham Pharmacia Biotech, Buckinghamshire, UK) in
accordance with the manufacturer’s instructions. Dilution levels for the serum samples were as follows: 1:1 for MMP-1,
1:250 for MMP-2, 1:1 for MMP-3, 1:20 for MMP-9, 1:150 for
TIMP-1, and 1:4 for TIMP-2. Dilution levels for the SF
samples were as follows: 1:400 for MMP-3 and 1:40 for
MMP-9. Levels of cartilage oligomeric matrix protein (COMP)
in the SF were measured by ELISA (AnaMar Medical, Uppsala, Sweden) in accordance with the manufacturer’s instructions. SF samples were diluted 1:40. Duplicate samples from
each individual were assayed.
Statistical analysis. Differences between groups were
analyzed using the Mann-Whitney U test, and analysis of the
matched pairs was performed using Wilcoxon’s signed-rank
test. Correlations between variables were assessed by Spearman’s
rank test. Bonferroni correction for multiple testing was applied
where indicated. A P value of less than 0.05 after Bonferroni
correction was considered to be statistically significant.
Figure 1. Immunohistochemical staining for matrix metalloproteinase
1 (MMP-1) (A), MMP-2 (B), MMP-3 (C), MMP-9 (D), tissue inhibitor
of matrix metalloproteinases 1 (TIMP-1) (E), and TIMP-2 (F) in the
lining layer as well as in the sublining layer. Synovial expression of
MMP-3 is characterized by cellular and diffuse interstitial expression
in the sublining layer and a less extensive expression in the lining layer.
In some patients, a characteristic distribution of interstitial expression
of MMP-3 adjacent to the lining layer is observed (G and H). MMP-9
is expressed primarily in the sublining layer of synovium, with a patchy
aspect that is especially pronounced in the perivascular regions, and
there is also staining of the intravascular cells (I and J). Original
magnification ⫻ 640 in A–F, H, and J, and ⫻ 320 in G and I.
Equal expression of MMPs and TIMPs in SpA
and RA synovium. Immunohistochemical staining of
synovial tissue samples from 41 patients with SpA
showed clear expression of MMP-1, MMP-2, MMP-3,
MMP-9, TIMP-1, and TIMP-2 in both the lining layer
and the sublining layer. As illustrated in Figure 1, the
different MMPs and TIMPs showed both a cellular and
an interstitial staining pattern, a more pronounced, but
Table 2. Correlation between synovial MMP and TIMP expression and local disease features in the synovium and synovial
fluid COMP levels in patients with spondylarthropathy*
cells present
Synovial fluid
COMP levels
* Values are Spearman’s rho correlation coefficients. Expression of matrix metalloproteinases (MMPs) and tissue inhibitors of
matrix metalloproteinases (TIMPs) was assessed by immunohistochemistry (semiquantitative scores on a 0–3 scale) and
correlated with local disease features (semiquantitative scores on a 0–3 scale). COMP ⫽ cartilage oligomeric matrix protein;
NS ⫽ not significant.
† P ⬍ 0.01.
‡ P ⬍ 0.05.
certainly not exclusive, staining for MMP-3 in the sublining layer than in the lining layer, and a pronounced
peri- and intravascular staining for MMP-9.
When both the degree and the pattern of immunoreactivity in the SpA samples were compared with
those from similar stainings of the 20 RA samples, we
found no significant differences for all of the investigated molecules (data not shown). However, within the
SpA group, the expression of MMP-1 was higher in the
AS and USpA synovial tissue samples (n ⫽ 22) than in
the PsA samples (n ⫽ 19) (P ⫽ 0.027 for the lining layer
and P ⫽ 0.010 for the sublining layer), and a similar
difference was observed within the SpA group in the
levels of MMP-2 in the sublining layer (P ⫽ 0.042). The
pattern of expression, however, was similar between the
AS/USpA patients and PsA patients.
Correlation between synovial MMP/TIMP expression and local features of synovitis in SpA. Because
of the strong expression of MMPs and TIMPs in SpA
synovitis and the potential role of this molecular system
in tissue remodeling, we investigated the relationship
between the expression levels of MMPs and TIMPs and
the features of local disease in SpA synovitis, such as
inflammatory cell infiltration (global number of inflammatory cells and number of polymorphonuclear cells in
the synovial membrane), extent of vascularization (num-
ber of blood vessels in the synovium), and degree of
cartilage breakdown (COMP levels in SF) (25,33). As
shown in more detail in Table 2, the expression of MMPs
3 and 9 and TIMPs 1 and 2 correlated with the level of
global inflammatory cell infiltration as well as with the
presence of polymorphonuclear cells. Moreover, the
expression of MMPs 2, 3, and 9 correlated with the
extent of vascularity, confirming the role of these MMPs
in vascular remodeling. Finally, SF levels of COMP, a
marker of collagen degradation, showed a strong inverse
correlation with the synovial expression of TIMP-2,
suggesting that high TIMP-2 levels are associated with
decreased cartilage breakdown in SpA. Of interest, the
COMP levels were equally elevated in the SF from RA
patients and the SF from SpA patients (median 26.2
units/liter, range 8.8–75.2 and median 26.4 units/liter,
range 12.4–90.0, respectively; P not significant).
Origination of serum MMP-3 from the inflamed
joint, reflecting peripheral synovitis in SpA. Since we
clearly demonstrated the expression of the MMPs and
TIMPs in inflamed SpA synovium, and since previous
reports have indicated that serum MMP levels are
elevated in SpA, we further investigated whether the
serum levels of MMPs and TIMPs were a reflection of
their expression in the peripheral joint. First, we evaluated the serum levels of MMPs and TIMPs in SpA
3.79 (1.90–53.59)
1,401.96 (325.59–2,933.64)
25.53 (0.08–1,190.64)†
447.36 (2.12–2,259.95)§
1,680.75 (972.95–2,892.36)
78.01 (7.20–541.65)
SpA patients
(n ⫽ 41)
4.35 (2.33–25.42)
1,791.19 (2.50–3,741.72)
44.59 (0.42–224.92)‡
570.15 (110.92–2,157.38)¶
1,774.44 (742.91–4,254.35)
79.89 (5.26–582.95)
RA patients
(n ⫽ 20)
5.23 (2.18–8.69)
1,149.53 (725.55–3,807.65)
10.61 (5.44–21.06)
237.92 (109.43–2,335.55)
1,642.88 (832.99–3,175.93)
51.22 (25.63–199.86)
Healthy controls
(n ⫽ 20)
4.3 (3.0–29.2)
1,336.6 (903.3–2,646.9)
89.4 (2.0–277.5)
301.2 (124.9–819.5)
1,474.1 (1,076.6–2,622.2)
57.2 (7.2–328.8)
4.6 (3.6–13.8)
1,545.2 (997.8–2,178.1)
9.8 (2.5–8.3)
383.6 (54.5–575.2)
1,609.4 (971.7–2,621.6)
31.3 (20.1–193.6)
After infliximab
SpA patients treated with infliximab (n ⫽ 12)
Levels of serum MMPs and TIMPs in SpA patients, RA patients, and healthy controls and in SpA patients before and after treatment with infliximab*
* Values for levels of biomarkers are the median (range) ng/ml. P values are by Mann-Whitney U test for comparisons between SpA patients, RA patients, and healthy controls,
and by paired Wilcoxon signed-rank test for comparisons before and after treatment with infliximab. See Tables 1 and 2 for definitions.
† P ⫽ 0.004 versus healthy controls.
‡ P ⬍ 0.001 versus healthy controls.
§ P ⫽ 0.090 versus healthy controls.
¶ P ⫽ 0.030 versus healthy controls.
Table 3.
Figure 2. Correlations between MMP-3 expression in the synovium, synovial fluid (SF), and serum of patients with spondylarthropathy (SpA). A
correlation is evident between MMP-3 levels in the SF (in ng/ml) and the expression of MMP-3 in the lining layer (semiquantitative score on a 0–3
scale) in SpA patients (n ⫽ 29; r ⫽ 0.497 by Spearman’s rank test, P ⬍ 0.01) (A), and also between SF MMP-3 and serum MMP-3 levels (both in
ng/ml) in SpA patients (n ⫽ 29; r ⫽ 0.567 by Spearman’s rank test, P ⬍ 0.01) (B). Serum MMP-3 levels were compared between ankylosing
spondylitis (AS) patients with exclusively axial involvement (n ⫽ 16) and AS patients with peripheral joint disease (n ⫽ 17) (C); significantly (P ⫽
0.009) higher serum MMP-3 levels are seen in AS patients with peripheral joint disease compared with those with exclusively axial symptoms. Boxes
and whiskers show the median and range. See Figure 1 for other definitions.
patients (n ⫽ 41) in comparison with RA patients (n ⫽
20) and healthy controls (n ⫽ 20). As shown in Table 3,
MMP-3 and MMP-9 were increased in the serum of SpA
and RA patients compared with healthy controls,
whereas there were no differences in the expression
levels of the other mediators. Furthermore, there were
no significant differences in the serum levels of MMPs
and TIMPs between the RA and SpA patients or
between the subtypes of SpA (data not shown).
On the basis of these results, we further analyzed
whether the expression of serum MMP-3 and MMP-9
originated from the inflamed peripheral joints of patients with SpA. ELISA analysis of SF from 41 SpA
patients revealed detectable levels of both MMP-3 (median 30,244.0 ng/ml, range 0–90,780.0) and MMP-9
(median 182.8 ng/ml, range 0–2,742.0). The SF levels of
MMP-3 correlated strongly with the expression of
MMP-3 in the lining layer (r ⫽ 0.497, P ⬍ 0.01) (Figure
2A), suggesting that secretion of MMP-3 occurs locally.
Moreover, the SF levels of MMP-3 were ⬃1,000-fold
higher than the MMP-3 levels in paired serum samples
(P ⬍ 0.001) and showed a strong correlation with the
serum MMP-3 levels (r ⫽ 0.567, P ⬍ 0.01) (Figure 2B).
Remarkably, we found significantly higher serum
MMP-3 levels in the AS patients who had peripheral
joint involvement (n ⫽ 16) (median 64.2 ng/ml, range
1.4–1,155.2) as compared with the AS patients who had
exclusively axial involvement (n ⫽ 17) (median 17.0
ng/ml, range 0–54.9) (P ⬍ 0.01) (Figure 2C). The
specificity of this finding was emphasized by the fact that
the parameters of systemic disease activity (C-reactive
protein [CRP] levels and erythrocyte sedimentation rate
[ESR]) did not differ between these 2 groups of AS
patients. In the same manner, serum MMP-3 levels in
the AS patients with exclusively axial involvement were
not significantly increased in comparison with those in
the healthy controls, and 6 of the 11 patients who had an
elevated ESR and increased CRP level demonstrated
serum MMP-3 levels in the normal range. In contrast, in
the AS patients with peripheral involvement, elevated
serum MMP-3 levels were observed in 10 of the 18
patients who had a normal ESR and in 4 of the 5 patients
with CRP values in the normal range (data not shown).
In contrast to the MMP-3 findings, SF levels of
MMP-9 were not significantly correlated with either the
synovial expression of MMP-9 or the serum levels of
MMP-9 (data not shown). Moreover, MMP-9 levels
were 3 times more elevated in the serum (median 447.4
ng/ml, range 2.1–2,260.0) than in the SF (median 182.8,
range 0–2,742.0) (P ⬍ 0.05). Finally, no differences in
serum MMP-9 levels could be demonstrated between
the AS patients with peripheral joint involvement (623.2
ng/ml, range 124.9–1,452.0) and those without peripheral joint involvement (953.8 ng/ml, range 556.0–
1,970.0; P not significant). The suggestion that the
expression of serum MMP-3, but not serum MMP-9, was
a reflection of peripheral joint inflammation rather than
global inflammation was further supported by the much
stronger correlation of SF and serum MMP-3 levels with
synovial inflammatory infiltration than with systemic
inflammatory parameters such as the CRP level and
ESR (data not shown).
Down-regulation of the MMP/TIMP system in
inflamed synovium by infliximab treatment. Since we
demonstrated the expression and involvement of the
MMPs/TIMPs in peripheral synovitis in SpA, we aimed
to confirm that this system was not constitutively expressed, but could be modulated by therapy, as suggested by microarray experiments during infliximab
treatment (data not shown). As shown in Table 1, there
was a significant and consistent improvement in the
parameters of global and peripheral joint disease in the
infliximab-treated group as compared with the placebotreated group.
Immunohistochemical analysis of synovial biopsy
tissue showed a significant down-regulation of MMP-3
(P ⫽ 0.017) and TIMP-1 (P ⫽ 0.026) and showed a
similar trend for MMP-9 (P ⫽ 0.059) in the lining layer
following treatment with infliximab. There was no treatment effect on MMP-1, MMP-2, and TIMP-2 within the
lining layer, which might be attributable to their low
baseline expression. Within the sublining layer, a significant down-regulation of synovial expression was observed for MMP-1 (P ⫽ 0.030), MMP-2 (P ⫽ 0.038),
MMP-3 (P ⫽ 0.023), MMP-9 (P ⫽ 0.011), TIMP-1 (P ⫽
0.011), and TIMP-2 (P ⫽ 0.010) following treatment
with infliximab (Figure 3).
Serum levels of MMP-1, MMP-2, MMP-9,
TIMP-1, and TIMP-2 from patients with peripheral
synovitis were not elevated at baseline as compared with
the baseline levels in healthy controls, and did not
change significantly after 12 weeks of treatment (Table
3). In contrast, serum levels of MMP-3 were elevated
prior to initiation of treatment (median 89.4 ng/ml,
range 2.0–277.5) and decreased substantially over 12
weeks (median 9.8 ng/ml, range 2.5–18.3) (P ⫽ 0.007) in
the infliximab-treated group (Figures 4A and B). In
comparison, the placebo-treated group showed a significant increase in serum TIMP-2 levels only (data not
shown). As demonstrated by sequential analysis at various time points between baseline and week 12, the
maximum down-regulation of serum MMP-3 levels in
patients with SpA and peripheral synovitis was achieved
within the first week of treatment and was sustained
during the whole followup period (Figure 4C).
Figure 3. Effect of infliximab (5 mg/kg intravenously at week 0, week 2,
and week 6) on synovial expression of MMP-1, MMP-2, MMP-3, MMP-9,
TIMP-1, and TIMP-2 in patients with SpA, as assessed by immunohistochemistry. Representative sections from the evaluations done at baseline
(left) and week 12 (12 we) (right) are shown (original magnification ⫻
320), and the corresponding semiquantitative scores on a 0–3 scale are as
follows: MMP-1, in the lining layer score 1 versus 0, in the sublining layer
score 2 versus 0; MMP-2, in the lining layer score 2 versus 1, in the
sublining layer score 2 versus 0; MMP-3, in the lining layer score 0 versus
0, in the sublining layer score 3 versus 0; MMP-9, in the lining layer score
1 versus 0, in the sublining layer score 2 versus 0; TIMP-1, in the lining
layer score 3 versus 0, in the sublining layer score 2 versus 0; TIMP-2, in
the lining layer score 1 versus 1, in the sublining layer score 1 versus 0. P
values are by paired Wilcoxon signed-rank test, comparing week 12 with
baseline values. See Figures 1 and 2 for definitions.
On the basis of preliminary findings obtained by
microarray, which revealed a decrease in synovial
Figure 4. Effect of infliximab (5 mg/kg intravenously at week 0, week 2,
and week 6) on serum levels of MMP-3 and MMP-9 in patients with SpA.
Serum levels of MMP-3 (A) and MMP-9 (B) for each individual patient
are illustrated at baseline (week [we] 0) and at week 12 after initiation of
therapy. In the placebo-treated patient cohort, no significant changes
were noted (data not shown). In addition, serum levels of MMP-3 were
evaluated at baseline, week 1, week 2, week 6, and week 12 after initiation
of therapy in 10 patients with SpA (C). A significant down-regulation of
serum MMP-3 is already evident at 1 week after initiation of the
infliximab treatment (ⴱ ⫽ P ⬍ 0.05). P values are by paired Wilcoxon
signed-rank test, comparing each time point with baseline. ns ⫽ not
significant (see Figures 1 and 2 for other definitions).
MMP-3 in SpA patients during infliximab treatment
(data not shown), the aim of the present study was to
investigate the MMP/TIMP system in SpA synovitis. The
present study demonstrated the presence of all investigated MMPs and TIMPs in both the lining and the
sublining layer of SpA synovium. Although both the
degree and the pattern of expression were specific to
each of the investigated MMPs and TIMPs, we found no
significant difference in the staining pattern between
SpA patients and a RA control group. Moreover, both
the level of expression and the staining pattern were
similar to those observed in previous studies in RA
Although the present study was not designed to
assess the complex functional interactions between
proenzymes, activated MMP forms, and inhibitory
TIMPs (38), the similar expression of the MMP/TIMP
system in SpA and RA might suggest that it is equally
involved in local disease features in both diseases.
Indeed, the demonstration of MMP-9 in intravascular
cells, in the vessel wall, and around the vessels, as well as
the correlations between the expression of MMPs 2, 3,
and 9 and the degree of vascularity in the present study
suggest that the previously demonstrated involvement of
these mediators in angiogenesis (39–42) also applies to
inflamed synovium in SpA. These findings are consistent
with those from a study indicating that elevated SF
MMP-9 concentrations in PsA were positively correlated
with the degree of synovial vascularization (43).
Another functional aspect of interest with regard
to MMPs and TIMPs is their role in cartilage and bone
degradation. Although it was not the primary aim of this
study to investigate this aspect in detail, it is interesting
to note that expression of TIMP-2 showed a strong
inverse correlation with the SF levels of COMP, a
marker of cartilage degradation. Nevertheless, the similar expression of all investigated MMPs and TIMPs in
SpA patients and RA patients might suggest that the
differences in destructive progression between these 2
diseases are not directly related to the MMP system,
although the complex functional regulation of the system precludes making strong conclusions from descriptive studies. It should be noted further that the PsA
patients included in the present study all fulfilled the
ESSG criteria for SpA and therefore might not be
representative of patients with polyarticular, erosive
These findings on the involvement of the MMP/
TIMP system in SpA synovitis and a previous report that
described elevated serum levels of MMP-3 in PsA
patients (44) raise the question as to whether serum
MMPs reflect the presence of peripheral joint disease in
SpA and thus might be useful biomarkers. When we
compared the serum concentrations in SpA patients who
had active peripheral joint disease with those in healthy
controls, we demonstrated elevated levels of MMP-3
with a similar trend for MMP-9 in the SpA patients, but
this was not found for the other investigated mediators.
Paralleling the data on synovial expression, the serum
levels of the different MMPs and TIMPs were not
different between SpA and RA patients, nor were they
different between the various SpA subgroups.
Further experiments involving the SpA samples
indicated that serum MMP-3, but not MMP-9, originated from the inflamed joint, since the serum concentrations were 1,000-fold lower than the SF levels and
correlated with the SF levels, which in turn correlated
with the synovial expression of MMP-3. This was further
confirmed by increased serum MMP-3 concentrations in
the AS patients with peripheral synovitis compared with
the AS patients with exclusively axial inflammation,
whereas the serum levels of MMP-9 as well as the CRP
levels and ESR were not different between the 2 AS
groups. Moreover, expression of serum, but not SF,
MMP-3 showed a weak correlation with the CRP levels,
but not with the ESR, whereas both the serum and the
SF MMP-3 levels correlated well with the degree of
inflammation of the synovium. Similarly, half of the AS
patients with exclusive axial involvement who had elevated CRP levels and an increased ESR demonstrated
MMP-3 serum levels in the normal range, whereas many
of the SpA patients with peripheral joint involvement
showed normal-range CRP levels and normal ESR
values but elevated levels of serum MMP-3. These data
are consistent with findings of increased serum MMP-3
levels in PsA patients but not AS patients (44) and with
a decrease in serum MMP-3 after total joint replacement (45).
Taken together, these findings indicate that serum MMP-3 reflects peripheral joint disease, rather than
global inflammation, in SpA patients. Thus, serum
MMP-3 warrants further evaluation for its value as a
biomarker of peripheral synovitis in clinical followup
and therapeutic trials. Possible explanations as to why
expression of serum MMP-3, but not MMP-9 or other
mediators, is a reflection of local inflammation remain
purely speculative.
As a way of approaching the value of these
biomarkers and to evaluate whether the MMP/TIMP
system in SpA is constitutive or can be modulated, we
investigated the effect of TNF␣ blockade by infliximab
on MMPs and TIMPs in synovium and serum. Previous
studies in RA indicated that synovial MMPs can be
down-regulated by methylprednisolone, methotrexate,
leflunomide, and interferon-␤ (46–48), while serum
MMPs are also decreased by TNF␣ blockade (49–51).
However, no significant effect of TNF␣ blockade has
been demonstrated on synovial MMP-1, MMP-3, and
TIMP-1 in RA (51) nor on serum MMP-1 and MMP-3
in AS (52), although the latest study might be biased by
the small number of patients with peripheral joint
The present study clearly indicated a rapid and
sustained down-regulation of serum MMP-3 after infliximab therapy, whereas there were no changes in any of
the other MMPs and TIMPs in either the infliximabtreated group or the placebo-treated group. This highly
significant decrease in serum MMP-3, which reflects the
clinical effect of infliximab on peripheral synovitis in
SpA (31,33), confirms the potential use of this agent for
monitoring synovial inflammation in clinical trials in
SpA. Moreover, the decrease in serum MMP-3 paralleled a strong down-regulation of the expression of
MMP-3 as well as the other MMPs and TIMPs in the
synovial membrane.
This study is the first to demonstrate the effect of
TNF␣ blockade on the MMP/TIMP system in the synovial membrane, and the findings are in accordance with
the stimulatory effect of TNF␣ in vitro on MMP and
TIMP production by synovial fibroblast and monocytes/
macrophages (16–18). However, it provides additional
evidence that whereas MMP production can by induced
by TNF␣ as well as numerous other inflammatory
stimulators in vitro, targeted blockade of TNF␣ is sufficient to profoundly down-regulate the MMP/TIMP system in inflamed SpA synovium in vivo. The major impact
of infliximab on the synovial expression of MMPs in SpA
supports the hypothesis that infliximab not only interferes with inflammation, but could also influence tissue
remodeling (vascularization, cartilage degradation) in
the joints of SpA patients (ref. 33, and Kruithof E, et al:
unpublished observations).
In conclusion, the present study indicates the
involvement of the MMP/TIMP system in the biologic
processes of peripheral SpA synovitis. Although synovial
tissue analysis is superior to serum analysis for the
evaluation of the MMP/TIMP system in inflammatory
arthritis, serum MMP-3 appears to be a specific biomarker for peripheral joint inflammation in SpA. Finally,
the major effect of infliximab on the MMP/TIMP system
warrants further prospective evaluation of the tissueremodeling effects of TNF␣ blockade in SpA, and raises
the possibility of using MMPs as potential therapeutic
targets in this disease.
The authors thank Virgie Baert for the excellent
technical contribution, and Ilse Hoffman and Bert Van der
Cruyssen for their assistance during the arthroscopy procedures.
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necrosis, factors, matrix, periphery, synovitis, spondylarthropathy, involvement, inhibitors, blockade, regulation, tumors, metalloproteinase
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