Synovial fluid exoglycosidases are predictors of rheumatoid arthritis and are effective in cartilage glycosaminoglycan depletion.код для вставкиСкачать
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: firstname.lastname@example.org. 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 188.8.131.52), ␤-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. 184.108.40.206), ␣ - D -mannosidase (EC 220.127.116.11), and ␤ - D galactosidase (EC 18.104.22.168) (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 22.214.171.124; Sigma-Aldrich) or ␤-D-N-acetyl-glucosaminidase (EC 126.96.36.199; 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. 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