Synovial fluid level of aggrecan ARGS fragments is a more sensitive marker of joint disease than glycosaminoglycan or aggrecan levels: a cross-sectional study

Abstract

Introduction: Aggrecanase cleavage at the 392Glu-393Ala bond in the interglobular domain (IGD) of aggrecan, releasing N-terminal 393ARGS fragments, is an early key event in arthritis and joint injuries. Here, we use a quantitative immunoassay of aggrecan ARGS neoepitope fragments in human synovial fluid to determine if this cleavage-site specific method better identifies joint pathology than previously available less specific aggrecan assays.

Methods: Synovial fluid (SF) from 26 people with healthy knees (reference) and 269 patients were analyzed in a cross-sectional study. Patient groups were acute inflammatory arthritis, acute knee injury, chronic knee injury and knee osteoarthritis (OA). Aggrecan ARGS fragments were assayed by ELISA using the monoclonal antibody OA-1. Total aggrecan content was analyzed by an ELISA using the monoclonal antibody 1-F21, and sulfated glycosaminoglycan by Alcian blue precipitation.

Results: Aggrecan ARGS fragment concentrations in all groups differed from the reference group (P < 0.001). The acute inflammatory arthritis group had the highest median level, 177-fold greater than that of the reference group. Median levels (in pmol ARGS/ml SF) were: reference 0.5, acute inflammatory arthritis 88.5, acute knee injury 53.9, chronic knee injury 0.5 and OA 4.6. In contrast, aggrecan and sulfated glycosaminoglycan concentrations varied much less between groups, and only acute inflammatory arthritis and acute knee injury were found to have a two-fold increase in median levels compared to the reference.

Conclusions: Levels of aggrecan ARGS fragments in human synovial fluid are increased in human arthritis, OA and after knee injury, likely reflecting an enhanced cleavage at the 392Glu-393Ala bond in the IGD by aggrecanase. An assay that specifically quantified these fragments better distinguished samples from joints with pathology than assays monitoring aggrecan or glycosaminoglycan concentrations. The newly developed ARGS fragment assay can be used to monitor aggrecanase activity in human joint disease and experimental models.

Figure 1

Anti-ARGS western blot of ELISA-captured material. Aggrecan fragments captured by the anti-keratan sulfate (KS)-coated plates were extracted after a completed ELISA and analyzed by western blot. Seventy-four wells of captured ARGS standards (STD) and 152 wells of SF from 152 patients were used. The samples were chondroitinase, keratanase, and keratanase II digested, separated on a SDS-PAGE gel, transferred to a polyvinylidene difluoride (PVDF) membrane and probed with the ARGS antibody OA-1. For comparison, the STD (0.5 μg sulfated glycosaminoglycan (sGAG)/well) and an SF D1 sample pooled from 40 osteoarthritis (OA) patients (0.75 μg sGAG/well) were used as controls. The size (kDa) and position of the molecular weight markers are indicated.

Figure 2

Regression analysis of aggrecan fragment data. The same samples of synovial fluid were analyzed by three different assays (see Material and Methods for details). Concentration of aggrecan fragments carrying the neoepitope ARGS by ELISA versus (a) sGAG concentration by Alcian blue precipitation (n = 293) and versus (b) aggrecan concentration by 1-F21 ELISA (n = 285). Solid lines show the first-order regression. Note the logarithmic X- and Y-axes. Spearman's rank order correlations (r_S) are given for each relationship with P < 0.0001.

Figure 3

Aggrecan fragment concentrations in the study groups. Concentrations of (a) ARGS fragments, (b) sulfated glycosaminoglycan (sGAG), and (c) aggrecan in the study groups healthy knee reference (REF), acute inflammatory arthritis (AA), acute knee injury (AI), chronic knee injury (CI), and knee osteoarthritis (OA). The boxes define the 25^th and 75^th percentiles with a line at the median, error bars defining the 10^th and 90^th percentiles and circles represents individual outliers. Note that in panel (a) the median level of the chronic injury group is the same as the lower limit of the box; 0.5 pmol ARGS/ml. After Bonferroni correction, P values below 0.013 are considered significant to retain the 0.05 overall significance level.

Figure 4

Aggrecan release after knee injury. Samples were ordered by time after knee injury (weeks) and by (a, b) meniscal injury alone (MEN) or (c, d) by anterior cruciate ligament rupture with or without an associated meniscus injury (ACL). Values are median concentrations of sulfated glycosaminoglycan (sGAG; open squares) and ARGS (filled squares) with 25^th and 75^th percentiles, compared with the medians (dashed lines) and 25^th and 75^th percentiles (shaded area) of the reference group on logarithmic Y-axes. Significant difference against the reference group at the 0.001 (***), 0.01 (**) and 0.05 (*) levels after Bonferroni correction is indicated.

Figure 5

Proportion ARGS of aggrecan in the study groups. The molar proportion of aggrecan fragments in synovial fluid (SF; measured by Alcian blue precipitation) detected as ARGS neoepitope fragments (measured by ARGS ELISA) in the study groups healthy knee reference (REF), acute inflammatory arthritis (AA), acute knee injury (AI), chronic knee injury (CI), and knee osteoarthritis (OA). The boxes define the 25^th and 75^th percentiles with a line at the median, error bars defining the 10^th and 90^th percentiles and circles represents individual outliers. After Bonferroni correction, P values below 0.013 are considered significant to retain the 0.05 overall significance level. Conversion from microgram sulfated glycosaminoglycan (sGAG)/ml to pmol aggrecan/ml was made assuming an average aggrecan molecular weight of 1.5 × 10⁶ g/mol and that 75% of this weight was sGAG.