Available online http://arthritis-research.com/content/8/4/R125 Available online http://arthritis-research.com/content/8/4/R125 y
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Nathalie Simard1, Gilles Boire2, Artur J de Brum-Fernandes2 and Yves St-Pierre 1INRS-Institut Armand-Frappier, University of Québec, Laval, Québec, Canada
2Division of Rheumatology, Department of Medicine, Centre hospitalier universita NRS-Institut Armand-Frappier, University of Québec, Laval, Québec, Canada pp
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Division of Rheumatology, Department of Medicine, Centre hospitalier universitaire de Sherbrooke, Université de Sher gy, Department of Medicine, Centre hospitalier universitaire de Sherbrooke, Université de Sherbrooke, Québec, Canad Corresponding author: Yves St-Pierre, yves.st-pierre@iaf.inrs.ca Corresponding author: Yves St-Pierre, yves.st-pierre@iaf.inrs.ca Received: 13 Apr 2006 Revisions requested: 24 May 2006 Revisions received: 13 Jun 2006 Accepted: 14 Jul 2006 Published: 19 Jul 2006
Arthritis Research & Therapy 2006, 8:R125 (doi:10.1186/ar2014)
This article is online at: http://arthritis-research com/content/8/4/R125 Received: 13 Apr 2006 Revisions requested: 24 May 2006 Revisions received: 13 Jun 2006 Accepted: 14 Jul 2006 Arthritis Research & Therapy 2006, 8:R125 (doi:10.1186/ar2014)
This article is online at: http://arthritis research com/content/8/4/R125 ,
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), 
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Despite decades of research, only a very limited number of
matrix metalloproteinase (MMP) inhibitors have been successful
in clinical trials of arthritis. One of the central problems
associated with this failure may be our inability to monitor the
local activity of proteases in the joints since the integrity of the
extracellular matrix results from an equilibrium between
noncovalent, 1:1 stoichiometric binding of protease inhibitors to
the catalytic site of the activated forms of the enzymes. In the
present work, we have measured by flow cytometry the net
proteolytic activity in synovial fluids (SF) collected from 95
patients with osteoarthritis and various forms of inflammatory
arthritis, including rheumatoid arthritis, spondyloarthropathies,
and chronic juvenile arthritis. We found that SF of patients with
inflammatory arthritis had significantly higher levels of proteolytic
activity than those of osteoarthritis patients. Moreover, the overall activity in inflammatory arthritis patients correlated
positively with the number of infiltrated leukocytes and the serum
level of C-reactive protein. No such correlations were found in
osteoarthritis patients. Members of the MMP family contributed
significantly to the proteolytic activity found in SF. Small-
molecular-weight MMP inhibitors were indeed effective for
inhibiting proteolytic activity in SF, but their effectiveness varied
greatly among patients. Interestingly, the contribution of MMPs
decreased in patients with very high proteolytic activity, and this
was due both to a molar excess of tissue inhibitor of MMP-1 and
to an increased contribution of other proteolytic enzymes. These
results emphasize the diversity of the MMPs involved in arthritis
and, from a clinical perspective, suggest an interesting
alternative for testing the potential of new protease inhibitors for
the treatment of arthritis. icity of protease activity changes were not directly measured. Indeed, conventional assays used to monitor the presence of
MMPs in SF, such as ELISA and zymography, do not provide
a direct measurement of their net proteolytic activity (NPA). The NPA depends on the activation status of the enzyme and
on the balance between active proteases and endogenous
protease inhibitors, such as tissue inhibitors of MMPs (TIMPs)
[7,8]. Hence, it is the equilibrium between active proteases
and inhibitors that determines the level of contribution of a
specific protease to cartilage degradation, and not simply its
expression level. This may explain why, while MMP-3 levels in
SF of rheumatoid arthritis (RA) patients are extremely high
[3,9], depletion of MMP-3 in animal models does not prevent ELISA = enzyme-linked immunosorbent assay; FASC = fluorescent-activated substrate conversion; FITC = fluorescein isothiocyanate; IA = inflam-
matory arthritis; IL = interleukin; MMP = matrix metalloproteinase; NPA = net proteolytic activity; OA = osteoarthritis; PBS = phosphate-buffered 
saline; RA = rheumatoid arthritis; SF = synovial fluids; TIMP = tissue inhibitor of MMP. Materials and methods
Reagents Gelatin and fluorescein isothiocyanate (FITC) were obtained
from Sigma (St Louis, MO, USA). Polystyrene microspheres
were purchased from Polysciences (Warrington, PA, USA). The blocking antibody specific for human MMP-9 was
obtained from Santa Cruz (Santa Cruz, CA, USA), and the
recombinant MMPs and their inhibitors were from Calbiochem
(San Diego, CA, USA). The human TIMP-1 ELISA kit was pur-
chased from R&D Systems (Minneapolis, MN, USA). Detection of matrix metalloproteinases by zymography
The gelatinase activity of SF was determined as previously
described [15]. The gelatinolytic activity was measured in arbi-
trary units by quantitative analysis of a negatively stained band
using computerized image analysis (Image Quant 5.0; Molec-
ular Dynamics, Sunnyvale, CA). Introduction Degradation of various macromolecules composing the extra-
cellular matrix is a hallmark of most forms of arthritis. These
changes are mediated by an excess of activity resulting from
an increased expression of the active form of the proteases,
and/or from an altered equilibrium between the proteases and
their inhibitors in inflamed synovial membrane and synovial flu-
ids (SF) [1-4]. This provided a rationale for the development of
broad-spectrum matrix metalloproteinase (MMP) inhibitors as
a new class of drugs [5,6]. The failure of these MMP inhibitors in clinical trials may at least
in part be explained by the fact that the magnitude and specif- Page 1 of 10
(page number not for citation purposes) Page 1 of 10
(page number not for citation purposes) Arthritis Research & Therapy    Vol 8 No 4    Simard et al. Arthritis Research & Therapy    Vol 8 No 4    Simard et al. stored at -80°C until used. SF were clarified by centrifugation
at 1500 × g for 15 min and were stored in aliquots at -80°C
for subsequent analysis. cleavage of aggrecan, nor does it prevent or reduce cartilage
destruction observed in specific forms of arthritis [10-12]. This lack of causal relationship between the expression levels
of specific MMPs and cartilage destruction may explain the
limited success of MMP inhibitors in clinical trials, and empha-
sizes the importance of measuring the NPA of proteases [13]. In the present work, using a flow-cytometric-based assay that
directly measures the NPA of MMPs in SF, we provide new
insights into the overall contribution of these enzymes to the
proteolytic activity in arthritic joints. The study included SF from patients with and without medica-
tion. Patients receiving medication were given nonsteroidal
anti-inflammatory drugs, either alone or in combination with
glucocorticosteroids (prednisone), or were given disease-
modifying antirheumatic drugs, such as methotrexate. This study was conducted with the approval of the ethics
review board of the Centre Hospitalier Universitaire de
Sherbrooke. Microsphere coating Polystyrene microspheres, 4.5 μm in diameter, were incubated
overnight at room temperature with the FITC-conjugated sub-
strate (15 μg/ml in PBS, pH 7.4) to allow noncovalent adsorp-
tion. The microspheres were then washed three times in PBS
(pH 7.4) containing 0.05% sodium azide. Microspheres were
kept at 4°C in PBS in the dark and were resuspended by gen-
tle vortexing before use. FASC assay and flow cytometric analysis Sampling of synovial fluids and sera The NPA was measured by fluorescent-activated substrate
conversion (FASC) [16] with the following modifications. Patients evaluated by rheumatologists from the Rheumatology
Division of the Centre Hospitalier Universitaire de Sherbrooke
were asked to participate in this study. Criteria for admission
to the study were the clinical indication for a therapeutic and/
or diagnostic arthrocentesis of one or several articulations and
a willingness to participate in the study. No exclusions were
made on any basis other than an inability or unwillingness to
give informed consent or the contamination of the SF by blood
during arthrocentesis. Fluorescent labelling of substrates
Denatured collagen was dissolved at a final concentration of 2
mg/ml in carbonate buffer (pH 9.2). FITC (dissolved in dimeth-
ylsulfoxide at 5 mg/ml) was added to the substrate (20 μl/mg). Labelling was carried out for 2 hours at room temperature. Free FITC molecules were removed by chromatography on
PD-10 columns (Pharmacia, Uppsala, Sweden) using PBS
(pH 7.4) as the eluent buffer. FITC is a small molecule, thereby
minimizing the steric hindrance around putative cleavage sites. It is also easily conjugated and compatible with most commer-
cial flow cytometers. A total of 95 samples of SF were used: 15 samples from
patients with established RA defined according to the 1987
American College of Rheumatology criteria [14], 14 samples
from patients with undifferentiated or recent-onset polyarthri-
tis, 26 samples from osteoarthritis (OA) patients, 13 samples
from patients with crystal-induced arthritis, and 27 samples
from patients with other noninfectious inflammatory arthritis
(IA), such as ankylosing spondylitis and reactive arthritis. A
patient with features of more than one rheumatic disease (e.g. OA and crystal-induced arthritis) was classified as having the
disease that clinically predominated at the time of
arthrocentrisis. Measure of net proteolytic activity in synovial fluids To quantitatively assess the actual level of proteolytic activity
in fluids, we used the FASC assay [16]. Fluorescent-labelled
large polypeptides derived from denatured collagen are immo-
bilized on polystyrene microspheres. These microspheres are
then incubated at 37°C for 18 hours with serial dilutions of SF,
and the proteolytic activity is monitored by the decrease of the
fluorescence emitted by the microspheres via flow cytometry. For the analyses, a two-parameter histogram with the forward-
angle light scatter and side scatter presented on the x axis and
the y axis, respectively, is used to position the window on sub-
strate-coated microspheres, thereby minimizing interference
with debris (Figure 1a, left panel). The NPA in the clinical sam-
ple is then obtained by measuring the mean fluorescence
intensity resulting from the loss of fluorescent substrate follow-
ing incubation of the microspheres with serial dilutions of fluids
at 37°C for 18 hours (Figure 1a, right panel). These results demonstrate that this approach can be used to
quantify the NPA resulting from the overall repertoire of pro-
teases present in biological fluids collected from human
patients. Available online http://arthritis-research.com/content/8/4/R125 Available online http://arthritis-research.com/content/8/4/R125 Available online http://arthritis-research.com/content/8/4/R125 pH values, we have chosen to measure the overall activity at
pH 7.4. ies have shown that large polypeptides derived from dena-
tured collagen are a convenient source of substrates to
measure the contribution of each of the four classes of pro-
teases, including serine, cysteine, and aspartic peptidases
[16,19] (data not shown). The enzymatic reactions were thus performed at 37°C for 18
hours in a final volume of 50 μl digestion buffer (20 mM Tris–
HCl, 150 mM NaCl, 10 mM Ca2+, 0.05% NaN3; pH 7.4). The
samples contained 10 μl SF at various dilutions and 5 × 104
(5 μl) FITC-labelled substrate-coated microspheres. The vol-
ume was completed to 50 μl with digestion buffer. The reac-
tion was stopped by adding 300 μl reading buffer (pH 9.5). To
titrate the proteolytic activity in arthritic SF, dilutions ranging
from 1:50 to 1:6250 (v/v) were prepared. Trypsin was used as
a positive control for enzymatic activity. In the first series of experiments, we quantitated the levels of
activity in SF from 95 patients with various forms of IA. To
accurately compare the net proteolytic activity between sam-
ples, the activity was determined by testing serial dilutions
(1:50–1:6,250); the activity is expressed as FASC units, 1
FASC unit being defined as the reciprocal dilution that
resulted in 40% cleavage of the fluorescent substrate. We
found that proteolytic activity was present at high levels in all
synovial samples, with IA patients exhibiting significantly
higher levels of NPA than OA patients (mean ± SEM, 2,589 ±
994 units/ml for IA versus 1,097 ± 810 units/ml for OA;
P < 0.001) (Figure 1b). Moreover, we also found that the titre
of activity varied greatly among the different samples, ranging
from approximately 300–500 units in most OA cases to more
than 3,500–5,000 units in SF obtained from the knee of
patients with spondyloarthropathies. Samples were analysed on a Coulter XL-MCL flow cytometer
(Hialeah, FL, USA) using standard optics for detection of FITC
fluorescence. For each sample, 2000 positive events were
analysed. Statistical analyses Data represent the mean ± standard error of the mean. The
statistical significance of differences between means was
determined by either Student's t test for comparisons between
two groups or Spearman rank order correlations. P < 0.05
was accepted as statistically significant. Interestingly, we also found that the NPA was similar in the flu-
ids collected from two different joints from the same patient on
the same day (Figure 1c), independently of the type of arthritis
and the level of proteolytic activity. This suggests that, in the
case of arthritis with involvement of several joints, systemic
factors play a dominant role in controlling the local proteolytic
activity. Finally, we did not detect any significant activity in nor-
mal SF obtained from healthy controls (<10 units/ml) (data not
shown). No enzymatic activity was similarly found in the serum
obtained from healthy subjects, or in serum of patients with
any form of arthropathies, consistent with previous reports
showing that human serum contains high concentrations of
endogenous protease inhibitors [20]. FASC assay and flow cytometric analysis After informed consent, venous blood (5 ml) and 2 ml SF col-
lected within 1 hour were sent to the appropriate clinical labo-
ratories. A differential blood cell count was performed on the
blood samples, and appropriate microbiologic studies to
exclude infectious arthritis, a differential nucleated cell count,
and crystal identification were performed on the SF. Venous
blood (10 ml) and SF in excess of the 2 ml needed for the clin-
ical purposes described were also collected. Sera were While the pH of normal synovial fluid is approximately 7.4, pre-
vious reports have shown that its value varies slightly in various
inflammatory conditions [17] – with a tendency to decrease
with conditions associated with an increasing leukocyte count
[18]. Since MMPs are neutral endopeptidases and are highly
active over a wide range of pH values, including lower acidic Page 2 of 10
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(page number not for citation purposes) Available online http://arthritis-research.com/content/8/4/R125 Proteolytic activity in synovial fluids of inflammatory 
arthritis patients correlates with the inflammatory state 
of the disease Extracellular proteases are secreted in SF by infiltrating inflam-
matory cells and/or by stromal cells stimulated by inflammatory
cytokines [21,22]. To assess whether inflammation does cor-
relate with the proteolytic activity in arthritic joints, we first
compared the NPA with the number of infiltrating leukocytes in
SF of IA patients. Our results showed that NPA correlated
positively (rs = 0.710, P < 0.001) with the number of infiltrated
leukocytes (Figure 2a) or with the number of infiltrated poly-
morphonuclear cells (rs = 0.696, P < 0.001) (Figure 2b). Weaker but significant correlations were also observed with The microspheres are analysed in a very small volume as they
pass through the flow cytometers' laser beam, and therefore
free fluorescent molecules (cleaved substrates) do not inter-
fere with the measure of fluorescence, obviating the require-
ment for washing the microspheres after incubation with the
samples prior to the flow cytometric data acquisition. This
approach therefore allows one to rapidly and accurately quan-
tify the presence of active extracellular proteases in human
body fluids upon cleavage of the peptide bond. Previous stud- Page 3 of 10
(page number not for citation purposes) Arthritis Research & Therapy    Vol 8 No 4    Simard et al. Figure 1 Figure 1
Proteolytic activity in synovial fluid of patients with osteoarthritis and inflammatory arthropathies
Proteolytic activity in synovial fluid of patients with osteoarthritis and inflammatory arthropathies. (a) Typical two-parameter histogram (left) with for-
ward-angle light scatter (FS) and side scatter (SS) (y and x axes, respectively), used to position the window on microspheres (one-parameter histo-
gram, middle) and to minimize the interference with debris. To determine the proteolytic activity in synovial fluids (SF), fluorescein isothiocyanate-
labelled polypeptides derived from denatured collagen (gelatin) were coated on polystyrene microspheres (shaded) and incubated with serial dilu-
tion of synovial fluid. LIN, Linear; LOG, Logarithmic. The mean fluorescent intensity at the top of each peak is then measured on 1,000–5,000 events 
(right). (b) Comparison of the level of proteolytic activity found in SF obtained from 26 osteoarthritis (OA) patients and 69 patients with inflammatory 
arthritis (IA), including patients with established rheumatoid arthritis (RA) or undifferentiated or recent-onset polyarthritis, patients with crystal-
induced arthritis, or patients with other noninfectious IA, such as ankylosing spondylitis and reactive arthritis. FASC, fluorescent-activated substrate 
conversion. (c) Proteolytic activity in SF collected from the left and right knees of arthritic patients on the same day. The expression level of MMP-9 is not indicative of the net 
proteolytic activity Excessive secretion of members of the MMP family is the hall-
mark of several inflammatory disorders, including arthritis. MMP-9, in particular, has been implicated in the degradation
and damage of articular cartilage in RA and OA [2-4]. We thus
examined the levels of MMP-9 in SF from 19 patients with dif-
ferent forms of arthritis, including RA, OA, chronic juvenile
arthritis, and other forms of arthritis, and compared them with
the levels of the NPA. The level of MMP-9 secretion was very
high in some fluids, although a significant heterogeneity was
observed among the samples (Figure 3a). These results were
not surprising since the levels of MMP-9 in SF have been
shown to vary according to several factors, such as the dis-
ease state, the local concentration of inflammatory cytokines,
and the number of infiltrating leukocyte subpopulations
[24,25]. Most importantly, we found that the level of MMP-9
did not correlate with the level of the NPA (Figure 3b). This
was particularly evident in samples 13, 20, 66, 95, and 98,
which had a very high level of NPA but very low levels of MMP-
9. 9. Available online http://arthritis-research.com/content/8/4/R125 Available online http://arthritis-research.com/content/8/4/R125 Figure 2
Correlation between the level of proteolytic activity found and the 
number of infiltrating leukocytes
Correlation between the level of proteolytic activity found and the 
number of infiltrating leukocytes. Positive correlation between the net 
proteolytic activity (NPA) and the number of (a) infiltrating leukocytes 
(rs = 0.710, P < 0.001) or (b) polymorphonuclear cells (PMN) (rs = 
0.696, P < 0.001) in synovial fluids of patients with inflammatory arthri-
tis. (c) No such correlation between the NPA and the number of infil- Figure 2 protein levels and the NPA was more striking (rs = 0.899,
P < 0.001) in patients with undifferentiated polyarthritis. In
contrast, no such correlation was observed between C-reac-
tive protein levels and the NPA in fluids of OA patients, or
between the NPA and sex or age of the patient (data not
shown). Proteolytic activity in synovial fluids of inflammatory 
arthritis patients correlates with the inflammatory state 
of the disease The results are representative of 
at least two independent experiments. GA, Gout arthritis. Proteolytic activity in synovial fluid of patients with osteoarthritis and inflammatory arthropathies
Proteolytic activity in synovial fluid of patients with osteoarthritis and inflammatory arthropathies. (a) Typical two-parameter histogram (left) with for-
ward-angle light scatter (FS) and side scatter (SS) (y and x axes, respectively), used to position the window on microspheres (one-parameter histo-
gram, middle) and to minimize the interference with debris. To determine the proteolytic activity in synovial fluids (SF), fluorescein isothiocyanate-
labelled polypeptides derived from denatured collagen (gelatin) were coated on polystyrene microspheres (shaded) and incubated with serial dilu-
tion of synovial fluid. LIN, Linear; LOG, Logarithmic. The mean fluorescent intensity at the top of each peak is then measured on 1,000–5,000 events 
(right). (b) Comparison of the level of proteolytic activity found in SF obtained from 26 osteoarthritis (OA) patients and 69 patients with inflammatory 
arthritis (IA), including patients with established rheumatoid arthritis (RA) or undifferentiated or recent-onset polyarthritis, patients with crystal-
induced arthritis, or patients with other noninfectious IA, such as ankylosing spondylitis and reactive arthritis. FASC, fluorescent-activated substrate 
conversion. (c) Proteolytic activity in SF collected from the left and right knees of arthritic patients on the same day. The results are representative of 
at least two independent experiments. GA, Gout arthritis. the number of monocytes (rs = 0.422, P < 0.001) and lym-
phocytes (rs = 0.405, P = 0.001) (data not shown). No such
correlation between the NPA and the number of infiltrating leu-
kocytes was observed in the case of patients with OA (Figure
2c). To further assess how the proteolytic activity correlates with
the inflammation, we examined C-reactive protein, a well-
known inflammatory marker [23]. We found that C-reactive
protein levels correlated positively (rs = 0.405, P = 0.009) with
the NPA in IA patients. The association between C-reactive To further assess how the proteolytic activity correlates with
the inflammation, we examined C-reactive protein, a well-
known inflammatory marker [23]. We found that C-reactive
protein levels correlated positively (rs = 0.405, P = 0.009) with
the NPA in IA patients. The association between C-reactive Page 4 of 10
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(page number not for citation purposes) Page 5 of 10
(page number not for citation purposes) Implication of matrix metalloproteinases in proteolytic 
reactivity found in synovial fluids of RA patients To further assess the contribution of MMP-9 and other mem-
bers of the MMP family in the NPA of patients with IA, we used
two hydroxamic acid-based broad-spectrum MMP inhibitors
(inhibitor II and inhibitor III). These inhibitors have been shown
to be selective for two relatively distinct repertoires of MMPs
– whereas inhibitor II inhibits MMP-1, MMP-3, MMP-7 and
MMP-9, inhibitor III blocks MMP-1, MMP-2, MMP-3, MMP-7
and MMP-13 [26]. All these MMPs are also elevated in SF during arthritis. For
instance, MMP-1, which is produced primarily by synovial
cells, and MMP-13, a product of the chondrocytes, have pre-
dominant roles because they are rate limiting in the process of
collagen degradation [27,28]. The efficiency of these two
inhibitors to block the enzymatic activity of MMPs at different
doses was first confirmed using recombinant MMPs (Figure
4a). When testing the activity using SF of patients with RA, we
found that while the percentage of inhibition of the NPA in the
presence of inhibitor III rarely exceeded 10–15%, the level of
inhibition obtained using inhibitor II was significantly higher,
ranging from 25% to 60% (Figure 4b). These results indicate
that MMPs contribute to a large extent to the NPA found in SF,
but that the repertoire of MMPs varies considerably among
patients. Correlation between the level of proteolytic activity found and the 
number of infiltrating leukocytes
Correlation between the level of proteolytic activity found and the 
number of infiltrating leukocytes. Positive correlation between the net 
proteolytic activity (NPA) and the number of (a) infiltrating leukocytes 
(rs = 0.710, P < 0.001) or (b) polymorphonuclear cells (PMN) (rs = 
0.696, P < 0.001) in synovial fluids of patients with inflammatory arthri-
tis. (c) No such correlation between the NPA and the number of infil-
trating leukocytes was observed in osteoarthritis (OA) patients. Each 
point represents one sample. The correlation was calculated using 
Spearman's rank correlation coefficient for each test. FASC, fluores-
cent-activated substrate conversion. Correlation between the level of proteolytic activity found and the 
number of infiltrating leukocytes
Correlation between the level of proteolytic activity found and the 
number of infiltrating leukocytes. Positive correlation between the net 
proteolytic activity (NPA) and the number of (a) infiltrating leukocytes 
(rs = 0.710, P < 0.001) or (b) polymorphonuclear cells (PMN) (rs = 
0.696, P < 0.001) in synovial fluids of patients with inflammatory arthri-
tis. Implication of matrix metalloproteinases in proteolytic 
reactivity found in synovial fluids of RA patients As a positive control, an aliquot of supernatant of HT1080 cell culture containing both MMP2 and MMP-9 
gelatinases was used. The results were obtained using duplicates and are representative of two independent experiments. Error bars represent the 
mean ± standard error of the mean. Zymographic analysis of MMP-2 and MMP-9 levels in arthritic synovial fluids
Zymographic analysis of MMP-2 and MMP-9 levels in arthritic synovial fluids. (a) Secretion of MMP-2 and MMP-9 was assessed by gelatin zymogra-
phy using standard procedures. Aliquots of 10 μl synovial fluids (SF) (diluted 1:10) were loaded into each lane. OA, osteoarthritis; snPA, seronega-
tive polyarthritis; rPA, seropositive rheumatoid polyarthritis; CPDD, calcium phosphate deposition disease; SpA, spondyloarthropathy; PA, 
polyarthritis; JCA, juvenile chronic arthritis; MA, microcrystalline arthritis. (b) Semiquantitative measures of the proMMP-9 level were carried out by 
densitometric analysis (scored as arbitrary units) and compared with the net proteolytic activity (NPA) found in the same SF. No correlations 
between the NPA and the level of the 225 kDa form of MMP-9 or with that of its active form (92 kDa) were observed (data not shown). FASC, fluo-
rescent-activated substrate conversion. As a positive control, an aliquot of supernatant of HT1080 cell culture containing both MMP2 and MMP-9 
gelatinases was used. The results were obtained using duplicates and are representative of two independent experiments. Error bars represent the 
mean ± standard error of the mean. Interestingly, however, we found that the effect of the MMP
inhibitor II dropped significantly (P = 0.046) as the NPA
increased (Figure 5a). We then examined whether the
decreased contribution of MMPs in those fluids with a high
proteolytic activity correlated with an increased TIMP-1 secre-
tion. Our results showed that these fluids had a very high level
of TIMP-1 (Figure 5b) that correlated positively with the
number of leukocytes (r = 0.910, P < 0.001) (Figure 5c), sug-
gesting that leukocytes may contribute positively to the forma-
tion of TIMP:MMP complexes. No such correlation between
TIMP-1 and the proteolytic activity was found in OA patients (r
= 0.880, P = 0.55). Implication of matrix metalloproteinases in proteolytic 
reactivity found in synovial fluids of RA patients (c) No such correlation between the NPA and the number of infil-
trating leukocytes was observed in osteoarthritis (OA) patients. Each 
point represents one sample. The correlation was calculated using 
Spearman's rank correlation coefficient for each test. FASC, fluores-
cent-activated substrate conversion. Page 5 of 10
(page number not for citation purposes) Arthritis Research & Therapy    Vol 8 No 4    Simard et al. Figure 3 Zymographic analysis of MMP-2 and MMP-9 levels in arthritic synovial fluids
Zymographic analysis of MMP-2 and MMP-9 levels in arthritic synovial fluids. (a) Secretion of MMP-2 and MMP-9 was assessed by gelatin zymogra-
phy using standard procedures. Aliquots of 10 μl synovial fluids (SF) (diluted 1:10) were loaded into each lane. OA, osteoarthritis; snPA, seronega-
tive polyarthritis; rPA, seropositive rheumatoid polyarthritis; CPDD, calcium phosphate deposition disease; SpA, spondyloarthropathy; PA, 
polyarthritis; JCA, juvenile chronic arthritis; MA, microcrystalline arthritis. (b) Semiquantitative measures of the proMMP-9 level were carried out by 
densitometric analysis (scored as arbitrary units) and compared with the net proteolytic activity (NPA) found in the same SF. No correlations 
between the NPA and the level of the 225 kDa form of MMP-9 or with that of its active form (92 kDa) were observed (data not shown). FASC, fluo-
rescent-activated substrate conversion. As a positive control, an aliquot of supernatant of HT1080 cell culture containing both MMP2 and MMP-9 
gelatinases was used. The results were obtained using duplicates and are representative of two independent experiments. Error bars represent the 
mean ± standard error of the mean. Zymographic analysis of MMP-2 and MMP-9 levels in arthritic synovial fluids
Zymographic analysis of MMP-2 and MMP-9 levels in arthritic synovial fluids. (a) Secretion of MMP-2 and MMP-9 was assessed by gelatin zymogra-
phy using standard procedures. Aliquots of 10 μl synovial fluids (SF) (diluted 1:10) were loaded into each lane. OA, osteoarthritis; snPA, seronega-
tive polyarthritis; rPA, seropositive rheumatoid polyarthritis; CPDD, calcium phosphate deposition disease; SpA, spondyloarthropathy; PA, 
polyarthritis; JCA, juvenile chronic arthritis; MA, microcrystalline arthritis. (b) Semiquantitative measures of the proMMP-9 level were carried out by 
densitometric analysis (scored as arbitrary units) and compared with the net proteolytic activity (NPA) found in the same SF. No correlations 
between the NPA and the level of the 225 kDa form of MMP-9 or with that of its active form (92 kDa) were observed (data not shown). FASC, fluo-
rescent-activated substrate conversion. Discussion This may
represent a sine qua non to achieve clinical success in the
development of new generations of protease inhibitors with
Our findings that the NPA is higher in RA than in OA are con-
sistent with previous reports that levels of MMPs and other
proteases are higher in RA [2,29,30]. This observation thus
supports the idea that inflammation plays an important role in
Figure 4
Effect of hydroxamate-based inhibitors on arthritic synovial fluids
Effect of hydroxamate-based inhibitors on arthritic synovial fluids. (a) 
Detection of the inhibitory effect of the matrix metalloproteinase (MMP) 
inhibitors (inhibitor II and inhibitor III) at different doses by fluorescent-
activated substrate conversion using recombinant human MMP-2 and 
MMP-9. Briefly, recombinant MMP-2 and MMP-9 were incubated with 
microspheres coated with fluorescein isothiocyanate-labelled polypep-
tides (derived from denatured collagen) at 37°C. The percentage of 
cleavage, measured by the loss of fluorescence, was measured by flow 
cytometry using standard optics for fluorescein (using a 525 nm band-
pass filter). (b) Effect of the hydroxamate inhibitors (used at 10 μM) on 
rheumatoid arthritis (RA) synovial fluids. Each bar represents one sam-
ple. The results were obtained using duplicates and are representative 
of two independent experiments. Error bars represent the mean ± 
standard error of the mean. Figure 5
Levels of TIMP-1 in synovial fluid of rheumatoid arthritis patients
Levels of TIMP-1 in synovial fluid of rheumatoid arthritis patients. (a) 
Inverse correlation between the matrix metalloproteinase (MMP)-
dependent proteolytic activity and the level of proteolytic activity in syn-
ovial fluids of rheumatoid arthritis (RA) patients (r = -0.521, P = 0.046). (b) Correlation between the level of TIMP-1 and the net proteolytic 
activity (r = 0.592, P = 0.07). (c) Positive correlation between the level 
of TIMP-1 and the number of infiltrated leukocytes (r = 0.910, P < 
0.001). The levels of TIMP-1 in synovial fluids were measured by com-
mercial ELISA. The contribution of MMPs to the proteolytic activity was 
carried out using the hydroxamate inhibitor II, as described in Figure 4. Each point represents one sample. The correlation was calculated 
using Spearman's rank correlation coefficient for each test. FASC, fluo-
rescent-activated substrate conversion. Figure 5 Figure 4 Figure 4            Effect of hydroxamate-based inhibitors on arthritic synovial fluids
Effect of hydroxamate-based inhibitors on arthritic synovial fluids. Discussion To the best of our knowledge, this is the first study assessing
the NPA in SF from patients with different forms of arthritis. Levels of active MMPs in the joint are frequently quantified
using immunological methods that do not discriminate
between MMPs that are active, that are in their latent form, or
that are bound to endogenous protease inhibitors. Our
approach clearly showed several results. First, the NPA in
patients with IA correlated positively with the number of infil-
trated leukocytes. Also, although the expression level of MMP-
9 is not indicative of the NPA found in SF, MMPs contribute to
a large extent to the proteolytic activity. Third, small molecular Page 6 of 10
(page number not for citation purposes) Available online http://arthritis-research.com/content/8/4/R125
Our findings that the NPA is higher in RA than in OA are con-
Figure 5
Levels of TIMP-1 in synovial fluid of rheumatoid arthritis patients
Levels of TIMP-1 in synovial fluid of rheumatoid arthritis patients. (a) 
Inverse correlation between the matrix metalloproteinase (MMP)-
dependent proteolytic activity and the level of proteolytic activity in syn-
ovial fluids of rheumatoid arthritis (RA) patients (r = -0.521, P = 0.046). (b) Correlation between the level of TIMP-1 and the net proteolytic 
activity (r = 0.592, P = 0.07). (c) Positive correlation between the level 
of TIMP-1 and the number of infiltrated leukocytes (r = 0.910, P < 
0.001). The levels of TIMP-1 in synovial fluids were measured by com-
mercial ELISA. The contribution of MMPs to the proteolytic activity was 
carried out using the hydroxamate inhibitor II, as described in Figure 4. Each point represents one sample. The correlation was calculated 
using Spearman's rank correlation coefficient for each test. FASC, fluo-
rescent-activated substrate conversion. Available online http://arthritis-research.com/content/8/4/R125 weight inhibitors were very effective in inhibiting MMP activity
in RA SF, but their effectiveness varied significantly among
patients. Finally, and most interestingly, increased TIMP-1
secretion by leukocytes in patients with IA shifted the balance
toward an MMP-independent proteolytic activity in those SF
with high NPA. Overall, these results stress the importance of
monitoring the repertoire of active proteases, not simply the
presence of proteases, in patients during the course of their
disease and under the influence of treatment. Discussion (a) 
Detection of the inhibitory effect of the matrix metalloproteinase (MMP) 
inhibitors (inhibitor II and inhibitor III) at different doses by fluorescent-
activated substrate conversion using recombinant human MMP-2 and 
MMP-9. Briefly, recombinant MMP-2 and MMP-9 were incubated with 
microspheres coated with fluorescein isothiocyanate-labelled polypep-
tides (derived from denatured collagen) at 37°C. The percentage of 
cleavage, measured by the loss of fluorescence, was measured by flow 
cytometry using standard optics for fluorescein (using a 525 nm band-
pass filter). (b) Effect of the hydroxamate inhibitors (used at 10 μM) on 
rheumatoid arthritis (RA) synovial fluids. Each bar represents one sam-
ple. The results were obtained using duplicates and are representative 
of two independent experiments. Error bars represent the mean ± 
standard error of the mean. Levels of TIMP-1 in synovial fluid of rheumatoid arthritis patients
Levels of TIMP-1 in synovial fluid of rheumatoid arthritis patients. (a) 
Inverse correlation between the matrix metalloproteinase (MMP)-
dependent proteolytic activity and the level of proteolytic activity in syn-
ovial fluids of rheumatoid arthritis (RA) patients (r = -0.521, P = 0.046). (b) Correlation between the level of TIMP-1 and the net proteolytic 
activity (r = 0.592, P = 0.07). (c) Positive correlation between the level 
of TIMP-1 and the number of infiltrated leukocytes (r = 0.910, P < 
0.001). The levels of TIMP-1 in synovial fluids were measured by com-
mercial ELISA. The contribution of MMPs to the proteolytic activity was 
carried out using the hydroxamate inhibitor II, as described in Figure 4. Each point represents one sample. The correlation was calculated 
using Spearman's rank correlation coefficient for each test. FASC, fluo-
rescent-activated substrate conversion. Levels of TIMP-1 in synovial fluid of rheumatoid arthritis patients
Levels of TIMP-1 in synovial fluid of rheumatoid arthritis patients. (a) 
Inverse correlation between the matrix metalloproteinase (MMP)-
dependent proteolytic activity and the level of proteolytic activity in syn-
ovial fluids of rheumatoid arthritis (RA) patients (r = -0.521, P = 0.046). (b) Correlation between the level of TIMP-1 and the net proteolytic 
activity (r = 0.592, P = 0.07). (c) Positive correlation between the level 
of TIMP-1 and the number of infiltrated leukocytes (r = 0.910, P < 
0.001). The levels of TIMP-1 in synovial fluids were measured by com-
mercial ELISA. The contribution of MMPs to the proteolytic activity was 
carried out using the hydroxamate inhibitor II, as described in Figure 4. Each point represents one sample. Discussion Since we found that MMP-2 is mostly found in its pro-
form in SF, our results favour the implication of MMP-13 Inter-
estingly, MMP-13, which is responsible for cleavage of type II
collagen [36,37], aggrecan [38], and fibrinogen [39], has
been shown to be increased in RA SF [3] and to be linked to
synovial inflammation and bone destruction [40]. Of course,
one has to be careful with such conclusion, as it is probable
that the repertoire of active proteases will vary with specific
subgroups of inflammatory arthritis. For instance, Peake and
colleagues recently showed that MMP-13 was not detected in
SF or serum of patients with juvenile idiopathic arthritis [41]. In the present article we have provided evidence that this
method constitutes a powerful tool to assess the performance
of enzyme inhibitors for therapeutic applications. For example,
our results with inhibitor II and inhibitor III suggest that MMP-2
and MMP-13 play a dominant role in the proteolytic activity
found in the SF of RA patients. One could thus be able to
determine which among the available protease inhibitors is
best suited to inhibit the proteolytic activity in SF of a given
patient. Since we found that MMP-2 is mostly found in its pro-
form in SF, our results favour the implication of MMP-13 Inter-
estingly, MMP-13, which is responsible for cleavage of type II
collagen [36,37], aggrecan [38], and fibrinogen [39], has
been shown to be increased in RA SF [3] and to be linked to
synovial inflammation and bone destruction [40]. Of course,
one has to be careful with such conclusion, as it is probable
that the repertoire of active proteases will vary with specific
subgroups of inflammatory arthritis. For instance, Peake and
colleagues recently showed that MMP-13 was not detected in
SF or serum of patients with juvenile idiopathic arthritis [41]. Our results showing elevated levels of TIMP-1 in SF with the
highest levels of NPA, both of which correlated with the levels
of leukocytes, further emphasize the importance of infiltrating
leukocytes in controlling the NPA of SF. Our results thus illus-
trate the need to measure the NPA resulting from the
(dis)equilibrium between active proteases and their inhibitors. Other workers have also reported a molar excess of TIMP-1 in
SF in animal models of OA, suggesting that matrix degenera-
tion in osteoarthritic joints could be promoted by other
proteases such as aggrecanase-1 and aggrecanase-2 [34]. Discussion The use of other protease inhibitors specific for cathepsins,
elastase, and other types of proteases previously found in SF
of RA patients [29,35] will be necessary to determine the con-
tribution of these proteases to the NPA in various diseases. We used the ability of the flow cytometer to accurately detect
different classes of particles, such as microspheres, based
upon a physical characteristic such as size and scattering, to
develop a simple and reliable method that allows qualitative
and quantitative measurements of specific enzymatic reaction
using fluorochrome-labelled substrate coated onto polysty-
rene microspheres [8]. The advantage of our approach is that
the fluorochrome-labelled substrates are used in the context of
laser flow cytometry, which increases the sensitivity of the
assay. Using spheres of multiple sizes, one could also use this
assay to perform real-time multiple determinations to deter-
mine the repertoire of different active proteases using selec-
tive substrates. Future work is clearly required to establish with more precision
the repertoire of active proteases in arthritic SF. The monitor-
ing of activity on a patient may allow adjusting the dosage of a
specific inhibitor or, alternatively, exploring whether additional
combinatorial treatments are required. From a clinical point of
view, therefore, this assay represents an ideal approach for
testing the potential of new protease inhibitors aimed at inhib-
iting disease progression. Failure to do so may explain at least
in part the current observation that MMP inhibitors failed in
clinical trials. It is probable, however, that other factors may
explain the failure of MMP inhibitors in clinical trials. For
instance, some members of the MMP family were recently
shown to exert an anti-inflammatory activity in some physiolog-
ical processes or diseases. This hypothesis received strong
support from data obtained using genetically engineered
MMP-deficient mouse models [42]. Moreover, cleavage of
chemokines by MMPs has been shown to generate chemokine
receptor antagonists that retain cellular binding affinity while
inhibiting the biological activity of the receptor [43]. SDF-1α
and other cytokines/chemokines can also be degraded by sev-
eral members of the MMP family that are constitutively
expressed at high levels in a particular tissue, impairing their
receptor-binding activity and their ability to mediate chemo-
taxis [44]. In contrast, other chemokines, such as IL-8, can be
proteolytically activated by MMP-9 and MT1-MMP [45]. Discussion The correlation was calculated 
using Spearman's rank correlation coefficient for each test. FASC, fluo-
rescent-activated substrate conversion. weight inhibitors were very effective in inhibiting MMP activity
in RA SF, but their effectiveness varied significantly among
patients. Finally, and most interestingly, increased TIMP-1
secretion by leukocytes in patients with IA shifted the balance
toward an MMP-independent proteolytic activity in those SF
with high NPA. Overall, these results stress the importance of
monitoring the repertoire of active proteases, not simply the
presence of proteases, in patients during the course of their
disease and under the influence of treatment. This may
represent a sine qua non to achieve clinical success in the
development of new generations of protease inhibitors with
superior clinical efficacy. Our findings that the NPA is higher in RA than in OA are con-
sistent with previous reports that levels of MMPs and other
proteases are higher in RA [2,29,30]. This observation thus
supports the idea that inflammation plays an important role in
controlling the protease/inhibitor ratio in SF [31], and is con- Page 7 of 10
(page number not for citation purposes) Arthritis Research & Therapy    Vol 8 No 4    Simard et al. enzymatic inhibitors in SF. This is a key issue as the degrada-
tion of the tissue architecture and disease state depend on the
biological activity of the enzymes; that is, on the ratio of free
active enzymes to inactive (inhibitor-bound) enzymes. sistent with the recent observation of a direct correlation
between tumour necrosis factor alpha/MMP production and
collagen degradation [32]. Indeed, we found that the NPA in
SF correlates positively with the number of infiltrating leuko-
cytes and with blood C-reactive protein levels. This finding
suggests that leukocytes control, either directly or indirectly,
the overall balance that results in the NPA found in SF – most
probably by secreting proteases, or by inducing the secretion
of proteases via the secretion of cytokines [33]. In the present article we have provided evidence that this
method constitutes a powerful tool to assess the performance
of enzyme inhibitors for therapeutic applications. For example,
our results with inhibitor II and inhibitor III suggest that MMP-2
and MMP-13 play a dominant role in the proteolytic activity
found in the SF of RA patients. One could thus be able to
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(page number not for citation purposes) Acknowledgements g
The authors thank Ms Marie Désy for statistical analysis. The work was 
supported by a grant from the Canadian Arthritis Network. YS-P and 
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