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Synovial fluid exoglycosidases are predictors of rheumatoid arthritis and are effective in cartilage glycosaminoglycan depletion.

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ARTHRITIS & RHEUMATISM
Vol. 48, No. 8, August 2003, pp 2163–2172
DOI 10.1002/art.11093
© 2003, American College of Rheumatology
Synovial Fluid Exoglycosidases Are Predictors
of Rheumatoid Arthritis and Are Effective in
Cartilage Glycosaminoglycan Depletion
Zsuzsanna Ortutay,1 Anna Polgár,2 Béla Gömör,3 Pál Géher,3 Tamás Lakatos,3 Tibor T. Glant,4
Renate E. Gay,5 Steffen Gay,5 Éva Pállinger,1 Csaba Farkas,6 Éva Farkas,1 László Tóthfalusi,1
Katalin Kocsis,1 András Falus,1 and Edit I. Buzás1
Objective. To analyze enzymes involved in joint
damage by simultaneous investigation of glycosidases
and matrix metalloproteinases (MMPs) in patients with
various joint diseases.
Methods. Activities of glycosidases ( ␤ - D glucuronidase, ␤-D-N-acetyl-glucosaminidase, ␤-D-Nacetyl-galactosaminidase, ␤-D-galactosidase, and ␣-Dmannosidase) were tested at an acidic pH as well as at
the original pH of the synovial fluid (SF) samples in
parallel with activities of MMP-1 and MMP-9.
Results. Patients with rheumatoid arthritis (RA)
were characterized by significantly elevated activities of
␤-D-glucuronidase and ␤-D-N-acetyl-glucosaminidase in
SF compared with patients with osteoarthritis, seronegative spondylarthritis, or acute sports injury. To select
the best predictor for distinguishing among patient
groups, a stepwise logistic regression analysis was performed; the strongest association was found to be
between RA and ␤ - D -glucuronidase/ ␤ - D -N-acetylglucosaminidase activities (measured at the pH of the
SF). Further, a significant correlation was observed
between the activity of SF ␤-D-N-acetyl-glucosaminidase
and the level of rheumatoid factor. In vitro digestion of
human hyaline cartilage samples revealed that the
dominant glycosidases, alone or in combination with
MMPs, proved to be effective in depleting glycosaminoglycans (GAGs) from cartilage.
Conclusion. These results suggest that exoglycosidases, which are present in the SF of RA patients, may
contribute to the depletion of GAGs from cartilage and
thereby facilitate the invasion of synovial cells and their
attachment to cartilage in RA.
In joint diseases, the major clinical symptoms and
disability of patients are caused by an irreversible destruction of hyaline cartilage. Enzymes capable of degrading extracellular matrix components (collagen and
aggrecan) and concomitantly exposing chondrocytes to a
variety of cytotoxic and/or apoptosis-inducing factors are
considered to be the major effector molecules in cartilage degradation.
Recently, significant advances have been made in
our understanding of joint destruction and the mechanism of proteolytic cleavage of cartilage. Active proteases are currently implicated in the destructive processes and include matrix metalloproteinases (MMPs),
the ADAM family (1), the ADAM-TS family (2), and
serine proteases (elastase, cathepsins, and granzymes)
(3–5). Of the 4 groups of MMPs, collagenase (MMP-1 in
particular) appears to be responsible for the degradation
of interstitial collagens. The gelatinases (including
MMP-2 and MMP-9) degrade the denatured form of
collagens, thus acting in synergy with MMP-1. The
stromelysins (including MMP-3) have a broader substrate specificity for non–connective tissue components.
Supported by grant T 032134 from the Hungarian Research
Foundation OTKA.
1
Zsuzsanna Ortutay, BS, Éva Pállinger, MD, Éva Farkas, BS,
László Tóthfalusi, MD, PhD, Katalin Kocsis, MD, PhD, András Falus,
PhD, Edit I. Buzás, MD, PhD: Semmelweis University, Budapest,
Hungary; 2Anna Polgár, MD: National Institute of Rheumatology and
Physiotherapy, Budapest, Hungary; 3Béla Gömör, MD, PhD, Pál
Géher, MD, PhD, Tamás Lakatos, MD: Polyclinic of Hospitaller
Brothers of St. John of God, Budapest, Hungary; 4Tibor T. Glant, MD,
PhD: Rush University at Rush–Presbyterian–St. Luke’s Medical Center, Chicago, Illinois; 5Renate E. Gay, MD, Steffen Gay, MD: University Hospital of Zurich, Zurich, Switzerland; 6Csaba Farkas, MD: Josa
András County Hospital, Nyı́regyháza, Hungary.
Address correspondence and reprint requests to Edit I.
Buzás, MD, PhD, Associate Professor, Department of Genetics, Cell
and Immunobiology, Semmelweis University, 4 Nagyvárad tér, H-1089
Budapest, Hungary. E-mail: buzedi@dgci.sote.hu.
Submitted for publication November 4, 2002; accepted in
revised form April 7, 2003.
2163
2164
ORTUTAY ET AL
Table 1. Characteristics of patients with various joint diseases
Rheumatoid arthritis
Seronegative spondylarthritis
Psoriatic arthritis*
Osteoarthritis
Acute joint injury
All patients
(n ⫽ 76)
Men
(n ⫽ 25)
Women
(n ⫽ 51)
Age, mean ⫾ SD
years (range)
Disease duration,
mean ⫾ SD months
31
16
12
15
14
5
9
6
2
9
26
7
6
13
5
53.6 ⫾ 3.2 (30–86)
46.2 ⫾ 11.7 (27–67)
46.4 ⫾ 9.8 (30–64)
67.8 ⫾ 15.9 (58–82)
42.9 ⫾ 11.6 (21–70)
79.3 ⫾ 138.6
53.1 ⫾ 60.8
66 ⫾ 47
99.3 ⫾ 135.1
⬍6 (after trauma)
* Patients with psoriatic arthritis are included in the group with seronegative spondylarthritis.
Members of the fourth group of the MMP family are not
secreted, but are membrane-type MMPs (MT-MMPs)
(6). MT-MMP-1 and MT-MMP-3 in particular have
been detected at sites of destruction in rheumatoid
arthritis (RA) (7,8). Several of these enzymes are currently considered therapeutic targets in arthritic diseases.
The family of glycohydrolases, however, has been
only marginally considered in arthritis research in the
last few decades. Although several groups of investigators reported elevated levels of glycosidases in rheumatic
diseases in the 1970s (9–11), those studies were not
pursued, and they have received little or no attention
lately. The lack of interest is surprising in light of the fact
that most cartilage matrix macromolecules are glycosylated, and some of them carry an extremely high
amount of carbohydrates. The carbohydrates, mostly
glycosaminoglycan (GAG) side chains (e.g., ⬃90% of
the molecular mass in aggrecan), may significantly affect
the proteolytic cleavage of the extracellular matrix
within the joints.
The present research addressed these considerations by investigating the effector mechanisms involved
in cartilage degradation in a more comprehensive way.
We did this by focusing both on glycosidases and on
MMPs implicated in the cartilage destructive pathways
and by providing evidence that glycosidases with high
activities present in the synovial fluid (SF) of patients
with RA are potent enzymes in the depletion of GAG
from hyaline cartilage. Based on reports in the literature
and on our results, we suggest that the hyaluronatedegrading exoglycosidases ␤-D-glucuronidase and ␤-D-Nacetyl-glucosaminidase facilitate cartilage destruction
in RA.
PATIENTS AND METHODS
Patients. Serum and SF samples from 62 patients (16
men, 46 women) treated at the National Institute of Rheumatology and Physiotherapy, Budapest, Hungary, were investigated. In addition, 14 patients (9 men, 5 women) undergoing
arthroscopy (subsequent to acute knee injury) at the Department of Orthopedics, University Medical School of Debrecen,
Debrecen, Hungary, were included in the study. The study was
approved by the local ethics committees, and all patients or
parents of children signed an informed consent form.
Patients included in the study had RA, seronegative
spondylarthritis (SNSA; 12 of whom had psoriatic arthritis
[PsA]), and osteoarthritis (OA) (Table 1). All RA patients met
the American College of Rheumatology (formerly, the American Rheumatism Association) 1987 revised criteria (12). All
PsA patients met the Moll-Wright criteria (13). All SNSA
patients had sacroiliitis; 4 of the SNSA patients (3 men, 1
woman) were suspected of having ankylosing spondylitis, but
they did not meet the New York criteria (14). OA patients did
not show any signs of inflammation; their erythrocyte sedimentation rates (ESRs) were ⬍30 mm/hour.
Demographic and clinical characteristics of the patients are also summarized in Table 1. Clinical and laboratory
records of patients included the number of swollen joints as
well as the platelet count, white blood cell (WBC) count,
serum rheumatoid factor (RF) level, and ESR.
Serum and SF samples. Blood and SF samples were
collected under sterile conditions and pelleted at 2,000 revolutions per minute for 20 minutes. Aliquots were stored at
⫺20°C until used.
Glycosidase assays. Enzyme activities were measured
in SF and serum samples using chromogenic substrates of
␤-D-glucuronidase, ␤-D-N-acetyl-glucosaminidase, ␤-D-N-acetylgalactosaminidase, ␣-D-mannosidase, and ␤-D-galactosidase:
p-nitrophenyl-␤-D-glucuronide, p-nitrophenyl-␤-D-N-acetylglucosaminide, p-nitrophenyl-␤-D-N-acetyl-galactosaminide,
p-nitrophenyl-␣-D-mannopyranoside, and p-nitrophenyl-␤-Dgalactopyranoside, respectively. All substrates were purchased
from Sigma-Aldrich (St. Louis, MO). Human SF samples (80
␮l) were diluted 1:1 either with sterile 0.15M NaCl solution in
deionized water or with 0.2M sodium acetate buffer (pH 5.8).
Next, 40 ␮l of 0.05M substrate was added to the samples (15).
The tubes were incubated at 37°C for 2 hours, and 500 ␮l
0.05M NaOH was added to each tube to terminate the enzyme
reactions.
Aliquots (200 ␮l) of each reaction tube were transferred to 96-well enzyme-linked immunosorbent assay plates
(MaxiSorp; Nunc Intermed, Copenhagen, Denmark), and optical densities (ODs) were measured at 405 nm by an MS
Reader (Multiskan MS; Labsystems, Helsinki, Finland). Enzyme activities were expressed as units, determined by using
different concentrations of enzymes with known activities: ␤-Dglucuronidase (EC 3.2.1.31), ␤-D-N-acetyl-glucosaminidase (EC
GLYCOSIDASES IN RHEUMATOID ARTHRITIS
2165
Figure 1. Comparison of enzyme activities in different patient groups. Bars show the mean and SEM.
Significant differences were found only between the rheumatoid arthritis (RA) and the non-RA patient
groups (ⴱ ⫽ P ⬍ 0.05). GLUC ⫽ ␤-D-glucuronidase; NAG ⫽ ␤-D-N-acetyl-glucosaminidase; OA ⫽
osteoarthritis; SNSA ⫽ seronegative spondylarthritis; SI ⫽ sports injury; MMP-1 ⫽ matrix metalloproteinase 1.
3.2.1.52), ␣ - D -mannosidase (EC 3.2.1.24), and ␤ - D galactosidase (EC 3.2.1.23) (all purchased from SigmaAldrich). All enzyme assays were performed under standardized conditions.
MMP activity. The activity of MMP-1 and MMP-9 in
SF samples was measured with Biotrak MMP-1 and MMP-9
activity assay systems (Amersham Pharmacia Hungary, Budapest, Hungary) according to the manufacturer’s instructions
(including activation of proMMP-1 and proMMP-9 by
APMA). Activities were compared with, and expressed as
ng/ml of, the active recombinant human enzymes (included in
the kits).
Histologic preparation for GAG staining. Human newborn cartilage samples were removed from patellar surfaces
during autopsy. Cartilage blocks (⬃2–3 mm3) were dissected,
and cartilage cubes were subjected to one of the following
enzymatic digestions in a 500-␮l volume at 37°C: 1) 0.187 units
of ␤-D-N-acetyl-glucosaminidase (Sigma-Aldrich) and 30 units
of ␤-D-glucuronidase (Sigma-Aldrich) for 2 hours, 2) 15 ng of
APMA-activated MMP-9 for 1 hour followed by 15 ng of
APMA-activated MMP-1 for 1 hour (both MMPs purchased
from Amersham Pharmacia Biotech) (samples digested as
described in 1 and 2 were incubated in 0.15M NaCl at 37°C for
an additional 2 hours), 3) digestion by MMPs as described in 2,
followed by procedure 1 (digestion by glycosidases), 4) glycosidase digestion 1 followed by MMP digestion 2, 5) incubation
in a 500-␮l volume of 0.15M NaCl without the addition of any
enzymes (for 4 hours at 37°C).
Tissue specimens were fixed in neutralized 4% formalin and embedded in paraffin, and 8–10-␮m sections were
stained with Safranin O (Sigma-Aldrich), a stain that binds
stoichiometrically to GAG (16). The images of the cartilage
sections were digitized using an Olympus DP50 camera (Olympus Optical, Hamburg, Germany). The superficial 1 mm–thick
layer of the articular cartilage was analyzed. Sections from 4
blocks of each treatment were analyzed (total of 20 sets of
measurements of each treatment). In each set of measurements, starting at the surface and moving perpendicularly
deeper down, articular cartilage was divided into ten 100-␮m ⫻
100-␮m quadrate areas. The ODs of these squares were
determined by Image-Pro Plus image analyzing software (Media Cybernetics, Silver Spring, MD). The OD of each square
was compared with the OD of the deepest layer (900–1,000
␮m) square of the same zone. The relative OD was determined
by subtraction. The mean relative OD of the 20 total sets of
measurements was calculated for each layer. Results are
expressed as the mean ⫾ SEM relative OD.
Detection of glycosidase-specific antibodies in SF samples. Nunc Immunoplates (MaxiSorp; Nunc Intermed) were
coated with ␤-D-glucuronidase (EC 3.2.1.31; Sigma-Aldrich) or
␤-D-N-acetyl-glucosaminidase (EC 3.2.1.52; Sigma-Aldrich)
(0.2 ␮g protein/well). Free binding capacity of the polystyrene
2166
ORTUTAY ET AL
Table 2. Comparison of glycosidase and matrix metalloproteinase (MMP) activities in synovial fluids
from patients with various joint diseases*
Enzyme
Full mode
GLUC (original pH)
NAG (original pH)
GLUC (pH 5.8)
NAG (pH 5.8)
MMP-1
MMP-9
Deviation
Degrees of
freedom
Residual
deviation
Mallows’ Cp
P†
15.831
3.681
0.325
0.088
0.427
0.100
31
30
29
28
27
26
25
44.36
28.52
24.84
24.52
24.43
24.00
23.90
46.34
32.49
30.79
32.45
34.35
35.90
37.79
0.000069
0.055036
0.568238
0.765952
0.513070
0.751537
* Stepwise logistic regression was used to select the optimal set of predictors to differentiate rheumatoid
arthritis (RA) patients from non-RA patients (those with osteoarthritis, seronegative spondylarthritis, or
acute knee injury). The information shown represents a slightly edited program output. The optimal
model has minimal Mallows’ Cp value, which in this case means that ␤-D-glucuronidase (GLUC) and
␤-D-N-acetyl-glucosaminidase (NAG), if measured at the original pH of the synovial fluid, may sufficiently
distinguish the RA group from the non-RA group. The significance of these two factors is confirmed by
the likelihood ratio test (chi-square test). The result is independent of using either forward or backward
elimination.
† By chi-square test.
surface was blocked with 200 ␮l of 1% bovine serum albumin
(Sigma-Aldrich). Based on preliminary experiments, 100 ␮l of
the serum and SF samples was tested at a 1:100 dilution. This
was followed by incubation with horseradish peroxidase–
conjugated rabbit anti-human polyvalent immunoglobulins
(Sigma-Aldrich) in 1:30,000 dilution. ODs were measured at
492 nm.
Flow cytometric monitoring of glycosidase activity.
␤-D-glucuronidase activity of peripheral blood cells was assessed by using the ImaGene Green GUS gene expression kit
(Molecular Probes, Eugene, OR) and adapting it for flow
cytometry using a FACSCalibur flow cytometer and a
CellQuest version 3.1 acquisition and analysis program (both
from BD Biosciences, San Jose, CA). After red blood cell lysis,
1 ⫻ 106 nucleated cells from heparinized blood samples were
incubated with 0.5 ␮l of ImaGene Green C12FDGlcU substrate, and fluorescence was detected at sequential time points
within the lymphocyte, monocyte, and granulocyte gates. Reaction specificity was confirmed by inhibition of the reaction by
preincubating samples with 1 ␮l of D-glucaric acid 1,4-lactone,
a ␤-D-glucuronidase inhibitor, for 30 minutes before addition
of the substrate. In some experiments, lymphocytes were
labeled with anti-human CD3–phycoerythrin (clone SK7) or
anti-human CD19–peridin chlorophyll protein (clone SJ25C1)
monoclonal antibodies (BD Biosciences), using (1 ␮g of
antibody/106 cells/100 ␮l of phosphate buffered saline (20
minutes) prior to addition of the fluorogenic glycosidase
substrate.
Statistical analysis. For statistical calculation, the
Splus6 for Windows software package (Insightful, Seattle,
WA) was used. Enzyme activities among patient groups were
compared using the honest significant difference method of
Tukey, and all possible comparisons were made. Tukey’s
method avoids the problem of multiple testing and was selected as the “best” approach by procedure “multicomp” of
Splus. Enzyme activities were also compared in terms of their
predictive power to distinguish the RA group from the
non-RA group. Stepwise logistic regression was applied for this
purpose.
RESULTS
Enzyme activities in SF samples from patients
with various joint diseases. A select group of exoglycosidases, including ␤-D-glucuronidase, ␤-D-N-acetylglucosaminidase, ␤-D-N-acetyl-galactosaminidase, ␣-Dmannosidase, and ␤-D-galactosidase, were tested in SF
samples using chromogenic substrates. Enzyme activities
were determined at both the original pH value of the
sample and at pH 5.8 in 4 disease groups: 31 patients
with RA, 16 with SNSA, 15 with OA, and 14 with acute
knee injury.
SF enzyme activities (mean ⫾ SEM ODs at the
original pH value of the sample and at pH 5.8, respectively), quite uniformly, were very low for ␤ - D galactosidase (0.017 ⫾ 0.0058 and 0.016 ⫾ 0.018),
␤-D-N-acetyl-galactosaminidase (0.039 ⫾ 0.021 and
0.047 ⫾ 0.028), and ␣-D-mannosidase (0.015 ⫾ 0.018 and
0.032 ⫾ 0.021). However, they were significantly higher
for ␤-D-glucuronidase (0.175 ⫾ 0.126 and 0.178 ⫾ 0.116)
and ␤-D-N-acetyl-glucosaminidase (0.116 ⫾ 0.14 and
0.2 ⫾ 0.208).
We therefore compared in subsequent experiments the activities of these two enzymes (␤-Dglucuronidase and ␤-D-N-acetyl-glucosaminidase) with
the levels of MMP-1 and MMP-9 in patients with various
joint diseases. As shown in Figure 1, ␤-D-glucuronidase
and ␤-D-N-acetyl-glucosaminidase activities (measured
GLYCOSIDASES IN RHEUMATOID ARTHRITIS
Figure 2. Discrimination between RA and non-RA patients according to GLUC and NAG activities. Each patient has a unique point
determined by his/her measured GLUC and NAG values. To visualize
the result of the stepwise logistic regression, the distributions of the patient groups are plotted on the GLUC–NAG plane. Patients without
RA are concentrated in the lower left corner. See Figure 1 for
definitions.
2167
at the original pH of the SF samples) were significantly
higher in RA patients than in patients with OA, SNSA,
or sports injury (P ⬍ 0.05). In contrast, no significant
intergroup difference was detected in the case of
MMP-1 or MMP-9 activity (Figure 1).
SF glycosidase activities as predictors of RA. To
select a parameter that clearly distinguishes patients
with RA from those with other joint diseases, a stepwise
logistic regression analysis was performed (Table 2). We
found that ␤ - D -glucuronidase and ␤ - D -N-acetylglucosaminidase activities, if measured at the original
pH of the SF, could serve as significant predictors for
RA. In contrast, neither the same enzyme activities
measured at pH 5.8 nor the levels of MMP-1 and
MMP-9 showed any difference (Table 2). To visualize
the results of the stepwise logistic regression, the distributions of the two types of patients were plotted on the
␤-D-glucuronidase and ␤-D-N-acetyl-glucosaminidase
plane. RA patient and non–RA patient groups were
clearly distinguished (Figure 2). RA patients were characterized by higher activities of both enzymes, while the
Figure 3. Scatterplots comparing enzyme activities across all patient groups. Straight lines correspond to
fitted linear regression lines. Corresponding correlation coefficients are shown. All correlations are
significant at a level of at least 0.05, but within-group correlations (between the two glycosidases or the two
MMPs) are stronger than correlations between enzymes of different groups. See Figure 1 for definitions.
2168
lower enzyme activity in the non-RA patients concentrated their distribution in the lower left corner of the
diagram (Figure 2).
Correlation of SF enzyme activities. We next
investigated correlations among activities of the two
major exoglycosidases of the SF samples ( ␤ - D glucuronidase and ␤-D-N-acetyl-glucosaminidase) and
those of MMP-1 and MMP-9. As shown in Figure 3, the
correlations between members of the different enzyme
groups (the exoglycosidases and the MMPs) were much
weaker (r ⫽ 0.39–0.52) than the within-group correlations (r ⫽ 0.79–0.90). The difference between the
between-group and within-group correlations suggests
that enzymes in the same group are regulated together,
but the groups themselves are regulated somewhat independently from each other.
We also made an attempt to find a significant
correlation of glycosidase activities with clinical and
laboratory parameters in RA patients (including the age
of the patient, the duration of the disease, the number of
swollen joints, the WBC and platelet counts, and the
ESR). The only significant correlation was confirmed
between the SF ␤-D-N-acetyl-glucosaminidase activity
and the RF level (Pearson’s product-moment correlation coefficient r ⫽ 0.819, P ⬍ 0.001).
Depletion of GAG from cartilage matrix. Our
next question concerned the relevance of our findings.
Thus, we examined whether the exoglycosidases ␤-Dglucuronidase and ␤-D-N-acetyl-glucosaminidase, which
had higher activities in SF from RA patients, were able
to induce the loss of GAG from cartilage. To demonstrate the degradation of cartilage by the enzymes,
human articular cartilage specimens were exposed to
both of these exoglycosidases and/or both MMP-1 and
MMP-9 (Figure 4). Safranin O was used to visualize the
depletion of GAG from the extracellular matrix. While
the dominant glycosidases proved to be effective in
depleting GAGs from cartilage, the most profound
effects (the lowest relative OD values) were seen to
result from the combined digestion procedure when
MMP digestion was followed by glycosidase treatment.
However, this combined digestion did not result in a
simple additive effect of the 2 enzyme groups. In a
control experiment, we excluded the possibility that
residual proteases could inactivate glycosidases (results
not shown). Therefore, we believe that a diffusiondependent depletion might have occurred during the
combined enzyme treatment.
The reduced depletion of GAGs in samples
treated with glycosidases first, followed by proteases, is
also most likely due to hindrance of diffusion in and out
ORTUTAY ET AL
Figure 4. Assessment of the depletion of glycosaminoglycan (GAG)
from human articular cartilage resulting from digestion with different
enzymes (visualized by Safranin O staining). A, Photomicrographs
show the result of digestion with ␤-D-glucuronidase and ␤-D-N-acetylglucosaminidase (glycosidases), the mixture of matrix metalloproteinases 1 and 9 (MMPs), or the combination of enzyme treatments
(MMPs followed by glycosidases [MMPs ⫹ Glyc.] or vice versa [Glyc.
⫹ MMPs]). A control specimen was incubated in 0.15M NaCl without
enzymes. Note the differences in the depths of the GAG-depleted
white zones. B, Relative optical densities (ODs) measured across the
GAG-depleted cartilage sections (computed by subtracting the OD of
100 ␮m ⫻ 100 ␮m squares at different distances from the surface from
the OD of the deepest layer of the sample, as described in Patients and
Methods). Values are the mean ⫾ SEM relative OD.
of the cartilage. Such hindrance could be related to
conformational changes of proteins upon interaction
with the negatively charged diffusion front of the digestion products of glycosidases.
Levels of circulating antibodies reactive with
glycosidases in serum and SF samples. The detection of
high enzyme activities in the SF of RA patients was
complemented by a search for antibodies against these
enzymes. Unexpectedly, we were able to detect anti–␤D-glucuronidase antibodies in serum and SF of certain
RA patients. The prevalence of the ␤-D-glucuronidase–
reactive antibodies (defined as the frequency [%] of
samples with a value above the mean ⫹ 2SD value of
OA samples) was 48.27% in serum and 21.74% in SF of
patients with RA and appeared to be related to the
reported anti–␤-D-glucuronidase perinuclear antineutrophil cytoplasmic antibodies in RA (17).
GLYCOSIDASES IN RHEUMATOID ARTHRITIS
2169
Figure 5. Flow cytometric monitoring of ␤-D-glucuronidase activities associated with peripheral blood lymphocytes,
monocytes, and granulocytes using a lipophilic substrate that diffuses freely across the membrane of viable cells. The
fluorescent enzyme cleavage products are retained by the cells. Fluorescence was detected 30, 60, and 90 minutes after
exposure to the substrate and was characterized according to the mean and SEM channel number. Arrowheads indicate
tested gates within scatterplots of blood cells. Patients with rheumatoid arthritis (RA; n ⫽ 5) (solid bars) are
distinguished from healthy controls (n ⫽ 4) (open bars) by higher ␤-D-glucuronidase activity. Significant differences between
RA patients and healthy controls are indicated. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01, by Mann-Whitney U test. X geo mean ⫽ average
of the logarithm of the channel numbers or linear values of the events within a marker, expressed as the anti-log.
Flow cytometric analysis of ␤-D-glucuronidase
activity of peripheral blood cells. To gain an insight into
the cell source of the elevated glycosidase activities, we
used a lipophilic substrate that diffuses freely across the
membrane of viable cells, thus enabling us to measure
␤-D-glucuronidase activity in the peripheral blood cells
by flow cytometry. The amount of fluorescent enzyme
cleavage products, retained by the cells, was clearly
higher in RA patients compared with the age- and
sex-matched healthy individuals (Figure 5). The difference was most pronounced for granulocytes, the cell
type that was associated with the highest ␤ - D glucuronidase activity, but significant differences were
also detected for monocytes and lymphocytes. Comparison of T (CD3⫹) and B (CD19⫹) lymphocytes in RA
patients did not reveal any significant difference in
cell-associated ␤-D-glucuronidase activity of the two
lymphocyte subsets. We failed to find a significant
correlation between SF- and lymphocyte-associated ␤-Dglucuronidase activities in the same RA patients (n ⫽ 4)
(data not shown), suggesting a limited relevance of
elevated lymphocyte-associated glycosidase activity values. However, our data reflect that patients with RA
have a generally elevated rate of glycosidase expression
in the cells of the systemic circulation.
DISCUSSION
Arthritis research, in general, has focused predominantly on the role of proteases in cartilage degradation. However, full understanding of the molecular
mechanisms of cartilage damage requires a more complex analysis of events, including evaluation of the role
of glycosidase enzymes. The present study identified two
dominant exoglycosidases in human SF samples: ␤-Dglucuronidase and ␤-D-N-acetyl-glucosaminidase. While
the activities of lysosomal glycohydrolases were traditionally assayed at acidic pH, the slightly basic pH of the
SF (18) compelled us to test enzyme activities at the
actual pH of the SF sample as well. This approach led to
the finding that the dominant glycosidases were highly
active at the original pH of the SF. When measured at
this pH, both ␤-D-glucuronidase and ␤-D-N-acetylglucosaminidase enzyme activities showed strong disease association with RA.
Substrate specificity of the dominant enzymes
identified them as being capable of fully degrading
hyaluronate by a stepwise alternating action. Indeed, the
viscosity of the SF is decreased in RA, and the most
evident explanation for this phenomenon is degradation
of hyaluronate by hydrolytic enzymes (19). Beyond
degradation of SF hyaluronate and the corresponding
decrease in lubricative properties, our findings are also
suggestive of a cartilage matrix–degrading capacity of
the above enzymes. This is consistent with a recent
report suggesting that hyaluronate degradation could
constitute an alternative mechanism of proteoglycan
release from cartilage (20). The 3 major mechanisms that
are considered to lead to hyaluronate degradation include
depolymerization by reactive oxygen radicals (21), cleavage
by hyaluronidases, and alternating cleavage by ␤-D-
2170
glucuronidase and ␤-D-N-acetyl-glucosaminidase. While
the catabolic contribution of ␤-D-glucuronidase and ␤-DN-acetyl-glucosaminidase was reported to be restricted
to hydrolysis of the oligosaccharides produced by the
action of hyaluronidase (22), the relative importance
of the exoglycosidases is strongly supported by their
joint specificity: ␤-D-glucuronidase and ␤-D-N-acetylglucosaminidase levels are higher in SF than in serum,
while hyaluronidase is characterized by an opposite
tissue distribution (9). Thus, exoglycosidases, presumably produced locally by the cells of the synovial membrane, might be important at the SF–cartilage or
pannus–cartilage interfaces.
The relevance of our findings to clinical disease is
strongly supported by the observed effectiveness of
␤-D-glucuronidase and ␤-D-N-acetyl-glucosaminidase in
depleting GAGs from cartilage, suggesting a possible
contribution of these enzymes to cartilage damage.
GAGs play a crucial role in the maintenance of compressive stiffness and resilience of hyaline cartilage. As a
result, GAG-depleted cartilage is highly vulnerable
when exposed to abrasive forces and gets worn off at the
surface upon loading. In several models of arthritis,
depletion of GAG from hyaline cartilage has been
demonstrated as an early histology finding (23). Our
glycosidase- or glycosidase/MMP-digested cartilage samples strongly resembled histology patterns seen in early
arthritis (showing a strikingly similar depletion of
GAG).
An important aspect of the question of the
significance of glycosidases in arthritis is the possible
interaction of proteases and glycosidases in cartilage
degradation. Recently, an increase in advanced glycation
end products was shown to result in decreased cartilage
degradation by MMPs (24). Thus, it has been suggested
that the level of cartilage glycation may influence the
progression of degradation. Conversely, protease action
can enhance tissue penetration and/or cleavage site
accessibility for glycosidases. Therefore, different enzyme groups could mutually render cleavage sites more
accessible to one another. It has been reported that
whenever an extensive aggrecan loss occurs (e.g., upon
stimulation by interleukin-1␤ [IL-1␤]), due to the proteolytic cleavage(s), there is a concomitant release of low
molecular weight hyaluronate.
MMP action has been implicated as the primary
event in cartilage degradation in association with GAG
release from the tissue (25). In concordance with this
concept, we found a massive loss of GAGs from the
superficial and middle layers of cartilage that had been
digested first with MMPs and next with glycosidases.
ORTUTAY ET AL
Since MMP-1 and MMP-9 are characterized by a predominantly collagenolytic activity, we propose that fragmentation of the type II collagen network facilitated the
tissue diffusion of exoglycosidases in our system. The
most probable mechanism of depletion of GAGs from
matrix by ␤-D-glucuronidase and ␤-D-N-acetyl-glucosaminidase is hyaluronate degradation (and subsequent
release of aggrecan components) supplemented with the
removal of terminal monosaccharides of GAGs (e.g.,
chondroitin sulfate).
Interpretation of the results raised the exciting
possibility that the high ␤-D-N-acetyl-glucosaminidase
activity that we measured in RA patients could have
been related to the action of a recently purified and
cloned nucleocytoplasmic enzyme (26,27) that is characterized by the same substrate specificity. However, this
possibility was ruled out by finding an acidic pH optimum for the ␤-D-N-acetyl-glucosaminidase in the SF
samples (data not shown). Also, the high correlation
between the activity of glucuronidase (a classic lysosomal enzyme) and glucosaminidase supports a common
lysosomal enzyme nature. Thus, it is more likely that the
high glucosaminidase activity in SF is related to or
identical to the activity of the lysosomal glycosidase
(hexosaminidase) that was recently reported to be the
dominant glycosidase of stimulated chondrocyte supernatants and RA sera (28,29).
What is the source of these enzymes in RA?
Chondrocytes have been implicated as an evident intrinsic cell source of MMPs (particularly in OA, in which
relatively few inflammatory cells are recruited, but also
in RA) (30). However, MMP-1 and MMP-9, two of the
IL-1– and tumor necrosis factor ␣–inducible MMPs
investigated in this study (31), are also known to be
secreted by inflammatory cells like neutrophil granulocytes (MMP-9) and monocyte/macrophages (MMP-1
and MMP-9) (32). Ligation of adhesive receptors (Lselectin and integrin CD11b/CD18 [Mac-1]) has been
shown to induce the release of MMP-9 from human
neutrophils, where it is stored in specific granules (33).
Also, differential production of MMP-9 by the synovial
membrane has been recently reported (34).
As far as glycosidases are concerned, while chondrocytes have been shown to release ␤-D-N-acetylglucosaminidase upon stimulation (35), recruited inflammatory cells in the joint cavity are the primary
candidates to release lysosomal glycosidases. The capacity for Ca2⫹-regulated exocytosis of lysosomal enzymes
has been shown for platelets, neutrophils, mast cells,
macrophages, cytotoxic T cells, and B cells (36–38). We
found higher ␤-D-glucuronidase activity in blood cells
GLYCOSIDASES IN RHEUMATOID ARTHRITIS
(particularly in granulocytes) of RA patients, which
supports the concept of the inflammatory cell origin of
the glycosidases.
The results presented here, by shedding light on
the possible interplay between proteases and glycosidases in RA, could facilitate further research in the field.
Analysis of the above enzyme systems is an important
complement to molecular and genetic studies in the effort
to fully understand the effector mechanisms of RA.
ACKNOWLEDGMENTS
The technical assistance of Zsuzsanna Vidra and Mercédesz Mazán is acknowledged.
2171
15.
16.
17.
18.
19.
20.
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