Changes in serum chondroitin sulphate epitopes 3-B-3 and 7-D-4 in early rheumatoid arthritis

J. Middleton, S. White, E. Parry, C. Jackson, J. Dixey and B. Ashton

Leopold Muller Arthritis Research Centre, Robert Jones and Agnes Hunt Orthopaedic and District Hospital, Oswestry, Shropshire, UK

Correspondence to: J. Middleton.


    Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Objectives. The aims of the present rheumatoid arthritis (RA) study were (1) to examine the levels of serum 3-B-3 and 7-D-4 to find out whether they are different from controls, (2) to find out whether the concentrations of these epitopes change with disease duration in early RA and (3) whether the serum concentrations of 3-B-3 and 7-D-4 in early RA are prognostic for subsequent disease progression.

Methods. The concentrations of 3-B-3 and 7-D-4 in sera were quantitated by immunoassays.

Results. The levels of 3-B-3 and 7-D-4 were significantly lower in RA than in controls (3- to 30-fold, P<0.001). Changes in 3-B-3 and 7-D-4 were apparent with disease duration. At first presentation, the 3-B-3 concentration was lowest and increased at 12 months (3-fold, P<0.001). This increase was transient since by 24 and 36 months the concentrations were not different to those at first presentation. The level of 7-D-4 was also lowest when the patients first presented at clinic and increased with time at 6 months (2-fold, P<0.001). The increase was more prolonged for 7-D-4, remaining elevated at 12, 24 and 36 months. The lack of correlations of serum 3-B-3 and 7-D-4 with clinical measurements showed that these markers were not prognostic for disease severity.

Conclusions. The levels of 3-B-3 and 7-D-4 differed between RA and control sera, and changed with disease duration. These markers were not prognostic in predicting disease outcome.

KEY WORDS: Chondroitin sulphate epitopes, 3-B-3 and 7-D-4, Early rheumatoid arthritis, Sera.


    Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Articular cartilage destruction is a feature of rheumatoid arthritis (RA) and leads to net loss of extracellular matrix molecules [1]. These breakdown products are released from the tissue, and enter the synovial fluid and the serum, where they are then cleared and excreted. Extracellular matrix molecules have been studied as potential markers of pathophysiological events that culminate in cartilage matrix destruction [24]. Aggrecan is the most abundant cartilage proteoglycan, comprising a protein core to which are attached chondroitin sulphate and keratan sulphate chains. In normal tissue, aggrecan binds to link protein and hyaluronan to form aggregates that are immobilized within the collagen fibril network.

Monoclonal antibodies 3-B-3 and 7-D-4 are specific for chondroitin sulphate and recognize neo-epitopes which may reflect pathophysiological events occurring in RA and osteoarthritis (OA) [5]. In dogs, these epitopes are absent or only weakly expressed in mature cartilage, and occur in very high levels in experimental OA cartilage [6, 7]. In addition, 3-B-3 and 7-D-4 occur in elevated levels in human articular cartilage from RA and OA joints, and in synovial fluid from knees following traumatic injury [3, 8]. It has been suggested that the increased expression of these epitopes may reflect an early reparative response to cartilage damage in which chondrocytes re-commence a high level of matrix synthesis similar to that seen in early development [8].

The aims of the present RA study were (1) to examine the levels of serum 3-B-3 and 7-D-4 to find out whether they are different from controls, (2) to find out whether the concentrations of these epitopes change with disease duration in early RA and (3) whether the serum concentrations of 3-B-3 and 7-D-4 in early RA are prognostic for subsequent disease progression.


    Subjects and methods
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Patients and controls
Patients were recruited at Oswestry as part of a nationwide early RA study (ERAS) and fulfilled the established American Rheumatism Association criteria for RA (Table 1Go). The 51 individuals were randomly chosen from a cohort of 120 patients with recent disease onset monitored at the Department of Rheumatology in Oswestry. Disease onset was defined as the time of onset of joint symptoms. Serum was taken when the patients first presented at clinic, and subsequently at 6, 12, 24 and 36 months. At each time point, clinical data were also recorded, including X-rays (Larsen grading), health assessment questionnaire (HAQ) as a functional index of joint mobility, grip strength, Ritchie index, pain score, duration of early morning stiffness and erythrocyte sedimentation rate (ESR).


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TABLE 1.  Details of patients and controls
 
For 3-B-3 and 7-D-4 groups, at presentation most patients were taking non-steroidal anti-inflammatory drugs (NSAIDs), either alone (49%) or in combination with analgesics or steroids (30%). The remaining patients were taking analgesics (9%) or steroids (4%) alone, or were not taking any medication (8%). Following first presentation, the majority of patients were administered second-line drugs so that by 6 months subjects were prescribed the following drugs alone or in combination with NSAIDs: sulphasalazine (55%), gold (16%) and methotrexate (2%). In addition, steroids (12%) and NSAIDs (10%) alone were taken, and some patients were without medication (4%). Similar proportions of drugs were in use at subsequent time points.

Control serum was obtained from volunteers employed at the Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, and residents in the local area. These subjects had no previous joint symptoms and were not on any current medication (Table 1Go).

Immunoassays
3-B-3 concentrations were measured in 51 patients and 7-D-4 levels were measured in the same serum samples from 35 patients. The 3-B-3 and 7-D-4 competitive ELISAs were as described by Hazell et al. [8] with the following modifications: in the 7-D-4 assay, bovine tracheal proteoglycan, kindly supplied by Dr M. Bayliss (Royal Veterinary College, London, UK) was used to coat the plates (3 µg/ml) and was the standard antigen (range 0.046–6 µg/ml). In both assays, the peroxidase substrate was tetramethyl benzidine (Sigma) diluted 1:4 in distilled water (100 µl). Colour development proceeded for 10 min and was stopped by the addition of 10% (1.8 M) sulphuric acid (50 µl). Absorbances at 450 nm were measured using a Bio-Tek EL 312 microplate reader.

Standards and samples were assayed in triplicate, the concentration of each epitope was calculated from the equation of the linear portion of the plot of inhibition of binding against concentration. Samples with epitope concentrations above the linear range were diluted with saline and reassayed. At least five samples were assayed in a minimum of four assays for the calculation of intra- and interassay variability. The performance characteristics of the assays are summarized in Table 2Go.


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TABLE 2.  Characteristics of immunoassays
 
Because of the high interassay variability, normal controls and patient samples were included in the same assays and all the time points from an individual patient were measured together.

Statistical analyses
The significance of differences between groups was calculated using the Mann–Whitney U-test for unpaired variables with Bonferroni correction for multiple tests. Correlations were calculated using the Spearman rank correlation coefficient or linear regression. The Fisher exact test was used to find out whether a significant proportion of patients showed increased epitope levels with time. P values of <0.05 were considered significant.


    Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The serum concentrations of the chondroitin sulphate epitope 3-B-3 in controls and RA patients are shown in Fig. 1AGo. The concentrations in control sera were higher than those of RA patients at any of the time points studied (P<0.001 in each case). The greatest difference in the medians was between the control and zero month RA (12-fold), and the least was between the control and 12 month RA (3-fold).



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FIG. 1.  (A) Concentrations of serum 3-B-3 with duration of rheumatoid arthritis (RA) and in controls. The median, 25th and 75th percentiles and range of the data are shown. The time points are from when RA patients first presented at clinic. *Significant difference (P<0.05) compared to control values. #Significant difference (P<0.05) compared to 0 month values. (B) Concentrations of serum 7-D-4 with duration of RA and in controls. The median, 25th and 75th percentiles and range of the data are shown. The time points are from when RA patients first presented at clinic. *Significant difference (P<0.05) compared to control values. #Significant difference (P<0.05) compared to 0 month values.

 
Patients when they first presented at clinic showed the lowest 3-B-3 concentration and this level did not change significantly at 6 months. At 12 months, the serum 3-B-3 levels had increased in 40 patients (80%, P<0.002), with a 3-fold increase in the median value compared to the zero time point (P<0.001). Thereafter, the 3-B-3 concentrations decreased so that the 24 and 36 month values were not significantly different to those at zero and 6 months.

The serum concentrations of 7-D-4 in controls and RA are shown in Fig. 1BGo. As with 3-B-3, the control values were higher than the RA patient values at each time point (P<0.001 in each case). The greatest difference in the medians was between the control and zero month RA (30-fold), and the least was between the control and 6 month RA (16-fold).

The lowest 7-D-4 level was evident when the patients first presented at clinic. At 6 months, there was a 2-fold increase in the median value (P<0.001) and an increase was shown by a significant proportion of patients (86%, P<0.005). At 12, 24 and 36 months, 7-D-4 concentrations, although elevated compared to the zero time point (P<0.02), were not different to the 6 month value.

There were no significant effects of age or gender on the amount of 3-B-3 and 7-D-4 in either the control or RA sera. The concentrations of the two epitopes showed no significant correlation to each other whether considered overall or at individual time points.

The association of levels of 3-B-3 and 7-D-4 in RA sera with clinical parameters was examined by regression analysis. There were no significant associations at any time point between the concentration of either antigen and the concurrent clinical parameters. In order to assess the potential usefulness of serum chondroitin sulphate epitopes as prognostic markers in RA, correlations were performed between zero, 6 and 12 month 3-B-3 and 7-D-4 concentrations and clinical variables at subsequent time points (up to 36 months). No significant correlations were found.

Serum creatinine concentrations did not differ significantly between controls and RA samples. In addition, there were no differences in serum creatinine levels between the various RA time points. These data suggest that the changes in the amounts of 3-B-3 and 7-D-4 with disease duration and in controls are not attributable to variation in renal clearance.


    Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The results of this study show that the concentrations of serum 3-B-3 and 7-D-4 epitopes are higher in control than early RA samples. The low level of epitope in RA may reflect inhibition of aggrecan synthesis, which could result from excessive production of inhibitory cytokines as part of the inflammatory process. In this context, high levels of tumour necrosis factor alpha and interleukin-1 are detectable in RA joints [911], and these cytokines have been shown to inhibit cartilage aggrecan synthesis in vivo and in vitro [1214]. However, as no information is available on epitope stability, one cannot rule out the possibility that low epitope levels in RA result from increased destruction of the epitope.

The large difference in 7-D-4 levels between control and RA sera is in agreement with that of knee synovial fluid where the 7-D-4 concentration is significantly lower in RA than controls [4]. This similarity suggests that the changes demonstrated in the serum reflect changes occurring in the synovial joints and that the 7-D-4 epitope detected in the serum derives, at least in part, from articular cartilage.

The difference between serum 3-B-3 in RA and controls was also evident, albeit smaller than that of 7-D-4 (3- to 12-fold for 3-B-3 as opposed to 16- to 30-fold for 7-D-4). However, in knee synovial fluid, Belcher et al. [4] found no difference between the concentration of 3-B-3 in RA and controls. The disparity between the reported synovial fluid 3-B-3 levels and those found by us in serum may reflect the fact that synovial fluid analysis monitors changes occurring in one joint, whereas the serum value reflects a contribution from all joints.

At the earliest time point, when patients first present at clinic, the levels of serum 3-B-3 and 7-D-4 were at their lowest, and over the next 6–12 months the concentrations of both markers increased. Although the changes in marker levels showed no significant association with clinical measures of disease activity and joint destruction, the increase corresponded to the time when the patients were administered second-line, `disease-modifying', drugs and thus may reflect the ability of the therapy to enable some attempt at cartilage repair to occur. In this context, anti-inflammatory drugs are reported to modulate proteoglycan synthesis and the development of cartilage erosions [1517].

Some differences were apparent between 7-D-4 and 3-B-3 with time. 7-D-4 levels increased significantly at 6 months, whereas 3-B-3 increased later at 12 months. In addition, the increase in 7-D-4 was more prolonged, remaining elevated at 12, 24 and 36 months. The increase in 3-B-3, however, was more transient since by 24 and 36 months the concentration had decreased to that of the zero time point. Differences are reported in the expression of the two epitopes in proteoglycans extracted from arthritic and normal human articular cartilage [3]. In addition, in synovial fluid, the difference between RA and control 3-B-3 and 7-D-4 levels varies (as mentioned above, [4]) and variation is reported following traumatic injury [8]. Taken together, these data suggest that subtle differences may exist in the expression and/or turnover of the two chondroitin sulphate epitopes.

It should be noted that serum levels of 3-B-3 and 7-D-4 could be affected by clearance from the lymph. Although this has not been documented in the case of chondroitin sulphate epitopes, it is known that some hyaluronan molecules from joint tissues enter the blood circulation via the lymphatic system [18]. Furthermore, the amount of hyaluronan transported via the lymph in RA patients could be elevated [19]. Disease-modifying drugs could also affect lymphatic activity and therefore influence the epitope levels in the blood.

The initial concentrations of serum 3-B-3 and 7-D-4 did not correlate with clinical data at subsequent time points. Therefore, over the time period studied, these markers do not appear to be useful as prognostic indicators of progressive RA. In this respect, other extracellular matrix constituents such as the aggrecan epitope 846, cartilage oligomeric matrix protein (COMP) and hyaluronan may be more effective [2, 20].


    Acknowledgments
 
We wish to thank Professor Bruce Caterson for donating 3-B-3 and 7-D-4 antibodies, Dr Cjilla Solymossy for Larsen scoring of X-rays, Sister Helen Dart for obtaining clinical data and Annabelle Darby for ELISA of control sera.


    References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 

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Submitted 11 August 1998; revised version accepted 6 April 1999.



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