385
SYNOVIAL FLUID ANALYSIS OF TWO GROUPS OF PROTEOGLYCAN EPITOPES DISTINGUISHES EARLY AND LATE CARTILAGE LESIONS TORE SAXNE and DICK HEINEGARD
Objective. To investigate whether fragmentation of proteoglycans in arthritis results in domains that have different levels of release from cartilage at different stages of the disease. Methods. Two regions of the proteoglycan, the hyaluronan-binding region and the glycosaminoglycanrich region of the core protein, were measured, by immunoassay, in knee joint synovial fluids of patients with rheumatoid arthritis or reactive arthritis. Results. Synovial fluid concentrations of the glycosaminoglycan-richregion were highest in rheumatoid arthritis patients who had little cartilage damage as determined by radiography, whereas release of the hyaluronan-binding region predominated in patients with advanced cartilage destruction. In reactive arthritis, release of the glycosaminoglycan-rich region predominated. Conclusion. These findings indicate that the hyaluronan-binding region is initially retained in the tissue during the development of cartilage destruction. The combined analysis of these markers offers a new From the Department of Rheumatology and the Department of Physiological Chemistry, Lund University, Lund, Sweden. Supported by the Medical Faculty of Lund, The Swedish Medical Research Council, Folksams Stiftelse, The German Rheumatism Research Centre, Berlin, Ciba-Geigy Corp., Alfred Osterlunds Stiftelse, Greta och Johan Kocks Stiftelser, Crafoords Stiftelse, Konung Gustav V:s Jubileumsfond, Lasarettets i Lund fonder, and Riksforbundet mot Reumatism. Tore Saxne, MD, PhD: Department of Rheumatology and Department of Physiological Chemistry; Dick Heineggrd, MD, PhD: Professor of Physiological Chemistry. Address reprint requests to Tore Saxne, MD, PhD. Department of Rheumatology, Lund University Hospital, S-221 85, Lund, Sweden. Submitted for publication June 21, 1991; accepted in revised form November 14. 1991. Arthritis and Rheumatism, Vol. 35, No. 4 (April 1992)
avenue for assessment of the degree of cartilage damage in arthritis.
Progressive destruction of articular cartilage is a prominent feature of rheumatoid arthritis (RA). A hallmark of the process is fragmentation of macromolecules by proteolytic cleavage and release of fragments into the synovial fluid (1). It has been shown, initially by staining for proteoglycans in the cartilage matrix, and later by synovial fluid analyses, that proteoglycans are degraded and released early in the course of the disease (2,3). Interestingly, it has also been shown that increased fragmentation and release of proteoglycans occurs during the acute phase of reactive arthritis (3,4). In the latter disease, identifiable tissue damage rarely develops, indicating that the mechanisms for repair are sufficient to prevent progressive destruction of matrix. This also emphasizes that cartilage involvement may occur in the absence of radiographic changes. We have previously shown that the release of proteoglycan from cartilage changes with matrix destruction, i.e., decreases when the cartilage mass diminishes (3). That study used an immunoassay with polyclonal antibodies with preferential specificity for epitopes in the chondroitin sulfate-rich region of the core protein of aggrecan, i.e., the cartilage-specific large, aggregating proteoglycan (5,6). From studies of cytokine-induced proteoglycan release, it appears that the proteoglycan may be cleaved such that part of it, the hyaluronan-binding region (HABr), remains bound to the hyaluronan and is therefore retained in the tissue (7,8). Thus, it is likely that fragments of aggrecan enriched in the HABr are released at a later stage
386
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Figure 1. a, lnhibition curves in the immunoassay for the hyaluronan-binding region (HABr), produced by human HABr (A), bovine HABr (B), and the core protein of human proteoglycan (C), in the quantities indicated. b, Inhibition curves in the immunoassay for the glycosaminoglycan-rich region of the proteoglycan core protein, produced by human HABr (A), the keratan sulfate-rich region of the human proteoglycan core protein (B), and the chondroitin sulfate-rich region of the human proteoglycan core protein (C), in the quantities indicated.
during cartilage destruction, compared with chon-
droitin sulfate-rich region fragments. If this is the case, it should be possible to grade the cartilage lesion in more detail by quantifying these different fragments found in synovial fluid. In the present study, we quantified HABr and glycosaminogl ycan-rich region core protein epitopes in synovial fluids of patients with RA or reactive arthritis, to elucidate whether these fragments of aggrecan show different patterns of release in different stages of the disease process, a n d whether their relative release differs with type of disease.
PATIENTS AND METHODS Patients. Synovial fluid samples from 83 patients with RA were obtained from the bank of synovial fluids stored at our institution. Fluids were selected according to the degree of radiographic knee joint damage, as determined by the Larsen score. With this scoring system, the degree of joint damage found radiographically is graded on a 6-point scale in which 0 denotes a normal joint and 5 the most advanced destruction (9). All patients fulfilled the established criteria for RA (10). Patients grouped according to Larsen score (group 0 comprising patients with a score of 0, group 1
comprising patients with a score of 1, etc.) did not differ with regard to age (median 63 years, range 34-79 years). The duration of synovitis at the time of arthrocentesis was significantly shorter in groups 0 and 1 (median 3 weeks, range 1 week-1 month) compared with the other groups (median 3 months, range 2 w e e k s 4 months) (P < 0.05). The disease duration was significantly shorter in groups 0, 1, and 2 compared with groups 3, 4, and 5 (median 2 years [range 6 months40 years] versus median 11 years [range 1-60 years]) (P < 0.05). All RA patients were treated with nonsteroidal antiinflammatory drugs (NSAIDs). The number of patients treated with oral glucocorticoids or slow-acting antirheumatic drugs was small and did not differ significantly among the RA groups. Synovial fluid samples were also obtained from 20 patients with reactive arthritis, defined according to the criteria of Aho et a1 (i.e., an acute-onset arthritis which develops soon after an infection elsewhere in the body, but in which viable microorganisms are not found in the joint tissues) (1 1). Five of these patients had arthritis following a Yersinia infection, 3 patients developed arthritis after a Salmonella infection, and in 4 patients, a Chlamydia infection preceded the onset of joint symptoms. In 8 patients, no precipitating infectious agent was found, but the clinical picture was compatible with a diagnosis of reactive arthritis. All 20 patients in this group had normal findings on radiography of the affected knee joint. Their median age at the time of arthrocentesis was 39 years (range 23-55 years). The
SYNOVIAL FLUID PROTEOGLYCAN EPITOPES median duration of synovitis was 1 week (range 1 day-1 month), and their median disease duration was 1 month (range 1 day-2 years). Twelve of these patients were treated with NSAIDs, and 8 did not receive any medication. The synovial fluid samples were collected, prepared, and stored as described previously (6). Immunoassays. Proteoglycan epitopes, mainly from the glycosaminoglycan-rich region, were measured by an inhibition enzyme-linked immunosorbent assay (ELISA) described previously (6). HABr fragments were measured by a novel ELISA utilizing antibodies that react selectively with the HABr. Goat polyclonal antiserum against the proteoglycan monomer (6) was chromatographed on a 1-ml affinity column of bovine HABr (3 mg) coupled to Sepharose 4B (Pharmacia, Uppsala, Sweden), by the cyanogen bromide procedure. HABr was prepared as described (12). The fraction of the antiserum that bound to the column, i.e., the material that should represent antibodies reacting with the HABr, was eluted with 3M potassium thiocyanate, 5 mM Tris, pH 7.0. Fractions showing absorbance at 280 nm were immediately dialyzed against 0.15M NaCI, 5 mM sodium phosphate, pH 7.4. An ELISA using these purified antibodies was then developed. Microfluor white plates (no. M119W; Dynatech, Alexandria, VA) were coated with bovine HABr (1 &ml) in O.05M sodium carbonate, pH 9.5. All procedures were performed at room temperature (22°C). After 24 hours, the plates were rinsed with 0.15M sodium chloride, 5 mM sodium phosphate, 0.05% (weightholume) Tween 20, pH 7.4, to remove unbound material. The plates were then coated with 2 mg/ml bovine serum albumin (Serva AG, Heidelberg, FRG) in coating buffer for 1 hour to reduce nonspecific binding, and were rinsed as above. Samples of synovial fluid (115 pl) diluted in 0.1M Tris, pH 7.5, were mixed with 115 pl of a 1 :600 dilution of the antiserum in 0.1M sodium chloride, O.OSM sodium phosphate, 0.05% Tween 20, pH 7.5. After preincubation for 24 hours, 200 pI of the mixture was added to the coated wells of the microtiter plate. After incubation for 1 hour, the plates were rinsed and 200 pI of a dilution of alkaline phosphataseconjugated rabbit anti-goat IgG (Sigma, St. Louis, MO) in 0.1M sodium chloride, O.05M sodium phosphate, 0.05% Tween 20, 2 mg/ml bovine serum albumin, pH 7.5, was added. After 1 hour, the plates were again rinsed, and 200 ~1 of the enzyme substrate, 2 pM methylumbellipherylphosphate (Sigma) in O.05M 2-amino-2-methyl- 1,3-propanediol, 0.05 mM MgCI,, pH 9.6, was added. Fluorescence was measured in a Perkin-Elmer (Beaconsfield, UK) Fluoroscence Spectrophotometer LS 5B equipped with a plate reader, immediately after substrate addition and again after 1 hour of incubation. A standard curve using bovine HABr, diluted as described above for the synovial fluid samples, was included in each microtiter plate. All samples were analyzed in triplicate, and the mean value was used for calculations. In experiments performed to demonstrate the specificity of the 2 antisera used, preparations of the keratan sulfate-rich region of the human core protein and of the human HABr prepared as described (12) were also used. Statistical analysis. Statistical calculations were performed using the Spearman correlation coefflcient or the
387
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Figure 2. Synovial fluid (SF) concentrations of the hyaluronanbinding region (HABr) of proteoglycan, in patients with rheumatoid arthritis or reactive arthritis. The rheumatoid arthritis patients were divided into 6 groups (groups 0-5) according to the degree of radiographically evident knee joint damage, as determined by the Larsen index (ref. 9). Horizontal bars indicate the median concentration in each group. See text for details.
Mann-Whitney U test (2-tailed) for comparisons between groups. P values less than 0.05 were considered significant.
RESULTS The affinity-purified antibodies reacted with both human and bovine HABr. In the inhibition ELISA, almost identical inhibition curves were produced with the HABr from the 2 species (Figure la). The antiserum showed some reactivity with the core protein, probably representing epitopes on the HABr (Figure la). In dilution experiments, we found that nonspecific factors in synovial fluid did not interfere in the assay (results not shown). A synovial fluid dilution of 1: 100 was used in subsequent studies of patient samples. The inter- and intraassay variation was < 10%. A separate experiment showed that human HABr, at the concentrations used in the assay, did not produce any inhibition in the ELISA for the glycosaminoglycan-rich region of the core protein (Figure 1b). Furthermore, the keratan sulfate-rich region of the human core protein did not produce any inhibition in this assay, which indicates that the antibodies
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patients with different degrees of radiographic joint damage, and in reactive arthritis patients versus RA patients, are further emphasized in Figure 4, where the calculated ratios of HABr to proteoglycan core protein concentrations in the different groups are shown. The ratios in RA patients increased with progression of joint damage (R, = 0.6537, P < 0.001). Reactive arthritis patients had the lowest ratio, indicating that in reactive arthritis, relatively more fragments containing glycosaminoglycan-rich region epitopes are released (P < 0.001 versus all other groups). Similar differences between the disease groups were found when the total synovial fluid content of the 2 groups of epitopes was compared, i.e., after correction for differences in volumes of aspirated fluid. The synovial fluid epitope levels did not correlate with duration of disease or synovitis. Statistical analysis of
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Figure 3. Synovial fluid (SF) concentrations of the glycosaminoglycan-rich region of the proteoglycan core protein (Proteoglycan), in patients with rheumatoid arthritis or reactive arthritis. The rheumatoid arthritis patients were divided into 6 groups (groups 0-5) according to the degree of radiographically evident knee joint damage, as determined by the Larsen index (ref. 9). Horizontal bars indicate the median concentration in each group. Note the logarithmic scale on the vertical axes. See text for details.
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preferentially recognize epitopes in the chondroitin sulfate4 ch region. Results of the synovial fluid measurements of the HABr and the proteoglycan core protein are shown in Figures 2 and 3, respectively. The HABr concentrations were distinctly higher in RA patients with Larsen scores of 3-5 than in RA patients with lower Larsen scores. A significant positive correlation between HABr concentrations and the Larsen index was found (R, = 0.5445, P C 0.001). In contrast, the proteoglycan core protein concentrations decreased with progression of cartilage damage (R, = -0.5299, P < 0.001). Reactive arthritis patients had synovial fluid concentrations of HABr as those found in the RA patients with little radiographic damage, but significantly higher concentrations of core protein ( P < 0.001). The different patterns of fragment release in RA
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Figure 4. Calculated ratios of the synovial fluid concentrations (conc.) of the hyaluronan-binding region (HABr) to the synovial fluid concentration of the glycosaminoglycan-rich region of the proteoglycan core protein (PG), in patients with rheumatoid arthritis or reactive arthritis. The rheumatoid arthritis patients were divided into 6 groups (groups 0-5) according to the degree of radiographically evident knee joint damage, as determined by the Larsen index (ref. 9). Horizontal bars indicate the median ratio in each group. Note the logarithmic scale on the vertical axes. See text for details.
SYNOVIAL FLUID PROTEOGLYCAN EPITOPES
389
the influence of treatment on the synovial fluid content of the 2 groups of epitopes was not possible in the RA patients, since the number of patients treated with glucocorticoids or slow-acting drugs was small and did not differ between the radiographic-score groups. The reactive arthritis patients treated with NSAIDs did not differ from those not treated, with respect to synovial fluid levels of the groups of proteoglycan antigens.
cation of cartilage damage as well as accurate assessment of the degree of cartilage damage at later stages, could prove valuable, for example, in selecting patients with similar degrees of cartilage involvement for controlled drug trials. Future development and improvement of such an assay should entail the use of monoclonal antibodies specific for select HABr and core protein epitopes.
DISCUSSION
ACKNOWLEDGMENTS
The pattern of release of 2 groups of epitopes of cartilage proteoglycan was found in this study to vary both between RA patients with different stages of cartilage involvement and between patients with this disease and those with reactive arthritis. The relative predominance of release of core protein epitopes, other than the HABr, early in the course of RA confirms observations reported previously (3). The relative predominance of a specific fragment, i.e., HABr, released at a later stage of the disease process has not been shown previously. This finding is consistent with the hypothesis that the proteoglycans are cleaved and those fragments not bound to hyaluronan are no longer retained in the tissue and are therefore released into the synovial fluid. The HABr is bound to hyaluronan in a manner that provides some protection from cleavage (13). It is therefore likely that it is released later in the process of cartilage breakdown than are regions of the core protein. The finding of relatively higher release of glycosaminoglycan-rich epitopes of the core protein in reactive arthritis further supports the notion that proteoglycan is released early in a disease process in which cartilage is affected. The present study shows for the first time that epitopes present on the same macromolecule, the proteoglycan, have different patterns of release, according to disease progression and disease type. Also, the data unambiguously show that released proteoglycans are fragmented. Interestingly, it appears that the proteoglycans released have been incorporated into the matrix, where the HABr remains, before degradation and release. It is also possible, however, that the HABr, if not bound to hyaluronan, becomes so degraded that it does not react in the assay. In this case, newly synthesized molecules could be released without having been incorporated into the matrix. The observations in this study should prove useful in further attempts to establish methods for “biochemical” grading of the cartilage lesion in arthritis. A sensitive method, which enables early identifi-
We are grateful to Mette Lindell and Karin Lindblom for their skillful technical assistance.
REFERENCES 1. Heineg%d D, Saxne T: Molecular markers of processes in cartilage in joint disease. Br J Rheumatol 30 (suppl 1):21-24, 1991 2. Edwards CG, Chrisman OD: Articular cartilage, The Scientific Basis of Orthopaedics. Edited by JA Albright, RA Brand. New York, Appleton-Century-Crofts, 1979 3. Saxne T, Heinegird D, Wollheim FA, Pettersson H: Difference in cartilage proteoglycan level in synovial fluid in early rheumatoid arthritis and reactive arthritis. Lancet i:127-128, 1985 4. Ratcliffe A, Doherty M, Maini RN, Hardingham TE: Increased concentrations of proteoglycan components in the synovial fluid of patients with acute but not chronic joint disease. Ann Rheum Dis 47:82&832, 1988 5. Heinegird D, Inerot S, Wieslander J, Lindblad G: A method for the quantification of cartilage proteoglycan structures liberated to the synovial fluid during developing degenerative joint disease. Scand J Clin Lab Invest 45:421-427, 1985 6. Saxne T, Heinegbrd D, Wollheim FA: Therapeutic effects on cartilage metabolism in arthritis as measured by release of proteoglycan structures into the synovial fluid. Ann Rheum Dis 45:491-497, 1986 7. Ratcliffe A, Tyler JA, Hardingham TE: Articular cartilage cultured with interleukin 1: increased release of link protein, hyaluronate binding region and other proteoglycan fragments. Biochem J 238571-580, 1986 8. Sandy JD, Neame PJ, Boynton RE, Flannery CR: Catabolism of aggrecan in cartilage explants: identification of a major cleavage site within the interglobular domain. J Biol Chem 266:8683-8685, 1991 9. Larsen A, Dale K, Eek M: Radiographic evaluation of rheumatoid arthritis and related conditions by standard reference films. Acta Radio1 Diagn 18:481491, 1977 10. Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS, Medsger TA Jr, Mitchell DM, N e w tadt DH, Pinals RS, Schaller JG, Sharp JT, Wilder RL,
390
Hunder GG: The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31:315-324, 1988 11. Aho K, Leirisalo-Rep0 M, Rep0 H: Reactive arthritis. Clin Rheum Dis 11:25-40, 1985 12. HeinegPrd D, Sommarin Y: Isolation and characterization of proteoglycans, Methods in Enzymology: Struc-
SAXNE AND
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tural and Contractile Proteins. D. Extracellular Matrix. Edited by LW Cunningham. Orlando, Academic Press, 1987 13. Heinegird D, Hascall V: The effects of dansylation and acetylation on the interaction between hyaluronic acid and the h yaluronic acid-binding region of cartilage proteoglycans. J Biol Chem 254:921-926, 1979
SYNOVIAL FLUID ANALYSIS OF TWO GROUPS OF PROTEOGLYCAN EPITOPES DISTINGUISHES EARLY AND LATE CARTILAGE LESIONS TORE SAXNE and DICK HEINEGARD
Objective. To investigate whether fragmentation of proteoglycans in arthritis results in domains that have different levels of release from cartilage at different stages of the disease. Methods. Two regions of the proteoglycan, the hyaluronan-binding region and the glycosaminoglycanrich region of the core protein, were measured, by immunoassay, in knee joint synovial fluids of patients with rheumatoid arthritis or reactive arthritis. Results. Synovial fluid concentrations of the glycosaminoglycan-richregion were highest in rheumatoid arthritis patients who had little cartilage damage as determined by radiography, whereas release of the hyaluronan-binding region predominated in patients with advanced cartilage destruction. In reactive arthritis, release of the glycosaminoglycan-rich region predominated. Conclusion. These findings indicate that the hyaluronan-binding region is initially retained in the tissue during the development of cartilage destruction. The combined analysis of these markers offers a new From the Department of Rheumatology and the Department of Physiological Chemistry, Lund University, Lund, Sweden. Supported by the Medical Faculty of Lund, The Swedish Medical Research Council, Folksams Stiftelse, The German Rheumatism Research Centre, Berlin, Ciba-Geigy Corp., Alfred Osterlunds Stiftelse, Greta och Johan Kocks Stiftelser, Crafoords Stiftelse, Konung Gustav V:s Jubileumsfond, Lasarettets i Lund fonder, and Riksforbundet mot Reumatism. Tore Saxne, MD, PhD: Department of Rheumatology and Department of Physiological Chemistry; Dick Heineggrd, MD, PhD: Professor of Physiological Chemistry. Address reprint requests to Tore Saxne, MD, PhD. Department of Rheumatology, Lund University Hospital, S-221 85, Lund, Sweden. Submitted for publication June 21, 1991; accepted in revised form November 14. 1991. Arthritis and Rheumatism, Vol. 35, No. 4 (April 1992)
avenue for assessment of the degree of cartilage damage in arthritis.
Progressive destruction of articular cartilage is a prominent feature of rheumatoid arthritis (RA). A hallmark of the process is fragmentation of macromolecules by proteolytic cleavage and release of fragments into the synovial fluid (1). It has been shown, initially by staining for proteoglycans in the cartilage matrix, and later by synovial fluid analyses, that proteoglycans are degraded and released early in the course of the disease (2,3). Interestingly, it has also been shown that increased fragmentation and release of proteoglycans occurs during the acute phase of reactive arthritis (3,4). In the latter disease, identifiable tissue damage rarely develops, indicating that the mechanisms for repair are sufficient to prevent progressive destruction of matrix. This also emphasizes that cartilage involvement may occur in the absence of radiographic changes. We have previously shown that the release of proteoglycan from cartilage changes with matrix destruction, i.e., decreases when the cartilage mass diminishes (3). That study used an immunoassay with polyclonal antibodies with preferential specificity for epitopes in the chondroitin sulfate-rich region of the core protein of aggrecan, i.e., the cartilage-specific large, aggregating proteoglycan (5,6). From studies of cytokine-induced proteoglycan release, it appears that the proteoglycan may be cleaved such that part of it, the hyaluronan-binding region (HABr), remains bound to the hyaluronan and is therefore retained in the tissue (7,8). Thus, it is likely that fragments of aggrecan enriched in the HABr are released at a later stage
386
SAXNE AND
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Figure 1. a, lnhibition curves in the immunoassay for the hyaluronan-binding region (HABr), produced by human HABr (A), bovine HABr (B), and the core protein of human proteoglycan (C), in the quantities indicated. b, Inhibition curves in the immunoassay for the glycosaminoglycan-rich region of the proteoglycan core protein, produced by human HABr (A), the keratan sulfate-rich region of the human proteoglycan core protein (B), and the chondroitin sulfate-rich region of the human proteoglycan core protein (C), in the quantities indicated.
during cartilage destruction, compared with chon-
droitin sulfate-rich region fragments. If this is the case, it should be possible to grade the cartilage lesion in more detail by quantifying these different fragments found in synovial fluid. In the present study, we quantified HABr and glycosaminogl ycan-rich region core protein epitopes in synovial fluids of patients with RA or reactive arthritis, to elucidate whether these fragments of aggrecan show different patterns of release in different stages of the disease process, a n d whether their relative release differs with type of disease.
PATIENTS AND METHODS Patients. Synovial fluid samples from 83 patients with RA were obtained from the bank of synovial fluids stored at our institution. Fluids were selected according to the degree of radiographic knee joint damage, as determined by the Larsen score. With this scoring system, the degree of joint damage found radiographically is graded on a 6-point scale in which 0 denotes a normal joint and 5 the most advanced destruction (9). All patients fulfilled the established criteria for RA (10). Patients grouped according to Larsen score (group 0 comprising patients with a score of 0, group 1
comprising patients with a score of 1, etc.) did not differ with regard to age (median 63 years, range 34-79 years). The duration of synovitis at the time of arthrocentesis was significantly shorter in groups 0 and 1 (median 3 weeks, range 1 week-1 month) compared with the other groups (median 3 months, range 2 w e e k s 4 months) (P < 0.05). The disease duration was significantly shorter in groups 0, 1, and 2 compared with groups 3, 4, and 5 (median 2 years [range 6 months40 years] versus median 11 years [range 1-60 years]) (P < 0.05). All RA patients were treated with nonsteroidal antiinflammatory drugs (NSAIDs). The number of patients treated with oral glucocorticoids or slow-acting antirheumatic drugs was small and did not differ significantly among the RA groups. Synovial fluid samples were also obtained from 20 patients with reactive arthritis, defined according to the criteria of Aho et a1 (i.e., an acute-onset arthritis which develops soon after an infection elsewhere in the body, but in which viable microorganisms are not found in the joint tissues) (1 1). Five of these patients had arthritis following a Yersinia infection, 3 patients developed arthritis after a Salmonella infection, and in 4 patients, a Chlamydia infection preceded the onset of joint symptoms. In 8 patients, no precipitating infectious agent was found, but the clinical picture was compatible with a diagnosis of reactive arthritis. All 20 patients in this group had normal findings on radiography of the affected knee joint. Their median age at the time of arthrocentesis was 39 years (range 23-55 years). The
SYNOVIAL FLUID PROTEOGLYCAN EPITOPES median duration of synovitis was 1 week (range 1 day-1 month), and their median disease duration was 1 month (range 1 day-2 years). Twelve of these patients were treated with NSAIDs, and 8 did not receive any medication. The synovial fluid samples were collected, prepared, and stored as described previously (6). Immunoassays. Proteoglycan epitopes, mainly from the glycosaminoglycan-rich region, were measured by an inhibition enzyme-linked immunosorbent assay (ELISA) described previously (6). HABr fragments were measured by a novel ELISA utilizing antibodies that react selectively with the HABr. Goat polyclonal antiserum against the proteoglycan monomer (6) was chromatographed on a 1-ml affinity column of bovine HABr (3 mg) coupled to Sepharose 4B (Pharmacia, Uppsala, Sweden), by the cyanogen bromide procedure. HABr was prepared as described (12). The fraction of the antiserum that bound to the column, i.e., the material that should represent antibodies reacting with the HABr, was eluted with 3M potassium thiocyanate, 5 mM Tris, pH 7.0. Fractions showing absorbance at 280 nm were immediately dialyzed against 0.15M NaCI, 5 mM sodium phosphate, pH 7.4. An ELISA using these purified antibodies was then developed. Microfluor white plates (no. M119W; Dynatech, Alexandria, VA) were coated with bovine HABr (1 &ml) in O.05M sodium carbonate, pH 9.5. All procedures were performed at room temperature (22°C). After 24 hours, the plates were rinsed with 0.15M sodium chloride, 5 mM sodium phosphate, 0.05% (weightholume) Tween 20, pH 7.4, to remove unbound material. The plates were then coated with 2 mg/ml bovine serum albumin (Serva AG, Heidelberg, FRG) in coating buffer for 1 hour to reduce nonspecific binding, and were rinsed as above. Samples of synovial fluid (115 pl) diluted in 0.1M Tris, pH 7.5, were mixed with 115 pl of a 1 :600 dilution of the antiserum in 0.1M sodium chloride, O.OSM sodium phosphate, 0.05% Tween 20, pH 7.5. After preincubation for 24 hours, 200 pI of the mixture was added to the coated wells of the microtiter plate. After incubation for 1 hour, the plates were rinsed and 200 pI of a dilution of alkaline phosphataseconjugated rabbit anti-goat IgG (Sigma, St. Louis, MO) in 0.1M sodium chloride, O.05M sodium phosphate, 0.05% Tween 20, 2 mg/ml bovine serum albumin, pH 7.5, was added. After 1 hour, the plates were again rinsed, and 200 ~1 of the enzyme substrate, 2 pM methylumbellipherylphosphate (Sigma) in O.05M 2-amino-2-methyl- 1,3-propanediol, 0.05 mM MgCI,, pH 9.6, was added. Fluorescence was measured in a Perkin-Elmer (Beaconsfield, UK) Fluoroscence Spectrophotometer LS 5B equipped with a plate reader, immediately after substrate addition and again after 1 hour of incubation. A standard curve using bovine HABr, diluted as described above for the synovial fluid samples, was included in each microtiter plate. All samples were analyzed in triplicate, and the mean value was used for calculations. In experiments performed to demonstrate the specificity of the 2 antisera used, preparations of the keratan sulfate-rich region of the human core protein and of the human HABr prepared as described (12) were also used. Statistical analysis. Statistical calculations were performed using the Spearman correlation coefflcient or the
387
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. . . . t
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Figure 2. Synovial fluid (SF) concentrations of the hyaluronanbinding region (HABr) of proteoglycan, in patients with rheumatoid arthritis or reactive arthritis. The rheumatoid arthritis patients were divided into 6 groups (groups 0-5) according to the degree of radiographically evident knee joint damage, as determined by the Larsen index (ref. 9). Horizontal bars indicate the median concentration in each group. See text for details.
Mann-Whitney U test (2-tailed) for comparisons between groups. P values less than 0.05 were considered significant.
RESULTS The affinity-purified antibodies reacted with both human and bovine HABr. In the inhibition ELISA, almost identical inhibition curves were produced with the HABr from the 2 species (Figure la). The antiserum showed some reactivity with the core protein, probably representing epitopes on the HABr (Figure la). In dilution experiments, we found that nonspecific factors in synovial fluid did not interfere in the assay (results not shown). A synovial fluid dilution of 1: 100 was used in subsequent studies of patient samples. The inter- and intraassay variation was < 10%. A separate experiment showed that human HABr, at the concentrations used in the assay, did not produce any inhibition in the ELISA for the glycosaminoglycan-rich region of the core protein (Figure 1b). Furthermore, the keratan sulfate-rich region of the human core protein did not produce any inhibition in this assay, which indicates that the antibodies
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patients with different degrees of radiographic joint damage, and in reactive arthritis patients versus RA patients, are further emphasized in Figure 4, where the calculated ratios of HABr to proteoglycan core protein concentrations in the different groups are shown. The ratios in RA patients increased with progression of joint damage (R, = 0.6537, P < 0.001). Reactive arthritis patients had the lowest ratio, indicating that in reactive arthritis, relatively more fragments containing glycosaminoglycan-rich region epitopes are released (P < 0.001 versus all other groups). Similar differences between the disease groups were found when the total synovial fluid content of the 2 groups of epitopes was compared, i.e., after correction for differences in volumes of aspirated fluid. The synovial fluid epitope levels did not correlate with duration of disease or synovitis. Statistical analysis of
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preferentially recognize epitopes in the chondroitin sulfate4 ch region. Results of the synovial fluid measurements of the HABr and the proteoglycan core protein are shown in Figures 2 and 3, respectively. The HABr concentrations were distinctly higher in RA patients with Larsen scores of 3-5 than in RA patients with lower Larsen scores. A significant positive correlation between HABr concentrations and the Larsen index was found (R, = 0.5445, P C 0.001). In contrast, the proteoglycan core protein concentrations decreased with progression of cartilage damage (R, = -0.5299, P < 0.001). Reactive arthritis patients had synovial fluid concentrations of HABr as those found in the RA patients with little radiographic damage, but significantly higher concentrations of core protein ( P < 0.001). The different patterns of fragment release in RA
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Figure 4. Calculated ratios of the synovial fluid concentrations (conc.) of the hyaluronan-binding region (HABr) to the synovial fluid concentration of the glycosaminoglycan-rich region of the proteoglycan core protein (PG), in patients with rheumatoid arthritis or reactive arthritis. The rheumatoid arthritis patients were divided into 6 groups (groups 0-5) according to the degree of radiographically evident knee joint damage, as determined by the Larsen index (ref. 9). Horizontal bars indicate the median ratio in each group. Note the logarithmic scale on the vertical axes. See text for details.
SYNOVIAL FLUID PROTEOGLYCAN EPITOPES
389
the influence of treatment on the synovial fluid content of the 2 groups of epitopes was not possible in the RA patients, since the number of patients treated with glucocorticoids or slow-acting drugs was small and did not differ between the radiographic-score groups. The reactive arthritis patients treated with NSAIDs did not differ from those not treated, with respect to synovial fluid levels of the groups of proteoglycan antigens.
cation of cartilage damage as well as accurate assessment of the degree of cartilage damage at later stages, could prove valuable, for example, in selecting patients with similar degrees of cartilage involvement for controlled drug trials. Future development and improvement of such an assay should entail the use of monoclonal antibodies specific for select HABr and core protein epitopes.
DISCUSSION
ACKNOWLEDGMENTS
The pattern of release of 2 groups of epitopes of cartilage proteoglycan was found in this study to vary both between RA patients with different stages of cartilage involvement and between patients with this disease and those with reactive arthritis. The relative predominance of release of core protein epitopes, other than the HABr, early in the course of RA confirms observations reported previously (3). The relative predominance of a specific fragment, i.e., HABr, released at a later stage of the disease process has not been shown previously. This finding is consistent with the hypothesis that the proteoglycans are cleaved and those fragments not bound to hyaluronan are no longer retained in the tissue and are therefore released into the synovial fluid. The HABr is bound to hyaluronan in a manner that provides some protection from cleavage (13). It is therefore likely that it is released later in the process of cartilage breakdown than are regions of the core protein. The finding of relatively higher release of glycosaminoglycan-rich epitopes of the core protein in reactive arthritis further supports the notion that proteoglycan is released early in a disease process in which cartilage is affected. The present study shows for the first time that epitopes present on the same macromolecule, the proteoglycan, have different patterns of release, according to disease progression and disease type. Also, the data unambiguously show that released proteoglycans are fragmented. Interestingly, it appears that the proteoglycans released have been incorporated into the matrix, where the HABr remains, before degradation and release. It is also possible, however, that the HABr, if not bound to hyaluronan, becomes so degraded that it does not react in the assay. In this case, newly synthesized molecules could be released without having been incorporated into the matrix. The observations in this study should prove useful in further attempts to establish methods for “biochemical” grading of the cartilage lesion in arthritis. A sensitive method, which enables early identifi-
We are grateful to Mette Lindell and Karin Lindblom for their skillful technical assistance.
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Hunder GG: The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31:315-324, 1988 11. Aho K, Leirisalo-Rep0 M, Rep0 H: Reactive arthritis. Clin Rheum Dis 11:25-40, 1985 12. HeinegPrd D, Sommarin Y: Isolation and characterization of proteoglycans, Methods in Enzymology: Struc-
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tural and Contractile Proteins. D. Extracellular Matrix. Edited by LW Cunningham. Orlando, Academic Press, 1987 13. Heinegird D, Hascall V: The effects of dansylation and acetylation on the interaction between hyaluronic acid and the h yaluronic acid-binding region of cartilage proteoglycans. J Biol Chem 254:921-926, 1979
