Destruction of articular cartilage by alpha 2 macroglobulin elastase complexes: role in rheumatoid arthritis

Abstract

Objective: Neutrophil elastase accounts for the ability of some fresh rheumatoid synovial fluids to degrade cartilage matrix in vitro. The aim of this study was to determine if enzyme activity could result from depletion of synovial fluid inhibitors or protection of the enzyme from inhibition.

Methods: The ability of synovial fluids to inhibit porcine pancreatic elastase was investigated together with chemical pretreatments capable of inactivating alpha 1 protease inhibitor (alpha 1PI) or preventing formation of alpha 2 macroglobulin (alpha 2M) elastase complexes. Subsequently, complexes of human neutrophil elastase with alpha 2M were prepared and applied to frozen sections of cartilage. Proteoglycan loss was quantified by alcian blue staining and scanning and integrating microdensitometry. Parallel studies were carried out using a low molecular weight chromogenic elastase substrate. The effects of alpha 1PI and SF on these systems were investigated. Finally, synovial fluids were subjected to gel filtration and the fractions assayed for elastase activity. High molecular weight fractions were pooled, concentrated, and tested for their ability to degrade cartilage sections.

Results: All synovial fluids reduced the activity of porcine pancreatic elastase, the inhibition mainly being attributable to alpha 1PI, whereas remaining activity resulted from complexes of elastase with alpha 2M. Complexes of human neutrophil elastase with alpha 2M were shown to cause proteoglycan degradation in frozen sections of human articular cartilage. Alpha 1PI prevented alpha 2M elastase complexes from degrading cartilage but not the chromogenic substrate. The data suggested that alpha 1PI does not inhibit elastase bound to alpha 2M but sterically hinders the complex. However, only one of five synovial fluids was able to completely block the actions of alpha 2M elastase complexes against cartilage. Gel filtration of rheumatoid synovial fluids showed elastase and cartilage degrading activity to be associated with fractions that contained alpha 2M, and not with fractions expected to contain free enzyme.

Conclusions: The data suggest that synovial fluid alpha 2M elastase complexes can degrade cartilage matrix in rheumatoid arthritis.

Figure 1

The elastase binding capacity of RA synovial fluid. Incubations were for 30 minutes at 37°C in the presence of methoxy-succinyl-ala-ala-pro-val-pNA (a chromogenic substrate). Open triangles = elastase activity of untreated synovial fluid. All other determinations were carried out with the addition of 1000 mU/ml porcine pancreatic elastase to each synovial fluid dilution. Solid triangles = elastase + synovial fluid. Open squares = elastase + synovial fluid pretreated with methylamine. Solid diamonds = elastase + synovial fluid pretreated with chloramine T. Solid squares = elastase + synovial fluid pretreated with both methylamine and chloramine T.

Figure 2

The effects of α₁PI (2 µM) on the ability of free human neutrophil elastase (NE) (68 nM) or elastase in the presence of α₂M (106 nM) to cleave either a low molecular weight chromogenic substrate or degrade cartilage matrix. To compare the two assays, data have been expressed as activity ratios (activity of elastase α₂M complexes/activity of free enzyme). Mean (SEM), n=5.

Figure 3

Proteoglycan loss loss from 4 µm cryostat sections of cartilage treated for 24 hours with 68 nM human neutrophil elastase (NE) in the presence of 106 nM α₂M. Also shown are the effects of five RA synovial fluids supernatants at 40% (v/v) on this degradation. Means (SEM), n=5.

Figure 4

Gel filtration of RA synovial fluid (solid squares) showing absorbance of fractions at 405 nm after 24 hour incubation with methoxy-succinyl-ala-ala-pro-val-pNA. Superimposed (open squares) is the gel filtration of a mixture of thyroglobulin (800 kDa) and carbonic anhydrase (29 kDa) for calibration purposes.