Effect of Leukocyte and Synovial Membrane Extracts on Cartilage Mucoprotein *

Abstract

The relatively simple composition of hyaline cartilage (1, 2) offers a unique opportunity to investigate the mechanism of erosion of this tissue in the presence of inflammatory synovitis. Shatton, Schubert and Malawista (1,2) have shown that about half of the dry weight of bovine nasal cartilage consists of chondromucoprotein material. A protein preparation of similar composition has been isolated from human rib cartilage (3). The other half of dried cartilage consists mainly of collagen (2). In the absence of evidence of an enzyme that can attack native collagen at physiological pH, it appeared logical to investigate the possibility that the primary process occurring during the erosion of cartilage was the degradation of the chondromucoprotein. Accordingly, the effect of extracts of leukocytes and inflamed synovial membrane on the chondromucoprotein was studied.Chondromucoprotein as extracted by Shatton and Schubert (1) contains about 70 per cent chondroitin sulfate and 30 per cent protein. It is water-soluble and highly viscous in solution. The latter property is in contrast to that of free chondroitin sulfate, which forms solutions of relatively low viscosity. On the basis of this difference, the rate of breakdown of the mucoprotein under the influence of cell-free extracts of leukocytes from the peripheral blood and of synovial membranes from patients with a variety of arthritides has been measured. Rapid decrease in viscosity on treatment with extracts of normal leukocytes and rheumatoid synovial membranes has been observed. Leukocyte extracts. Pellets of leukocytes obtained from the peripheral blood of normal subjects and of hospitalized patients with a normal peripheral blood picture were prepared by the addition of 1 vol of 6 per cent dextran solution to 3 vol of heparinized blood distributed in 15 ml conical centrifuge tubes. After allowing them to stand for 1 hour at 370 C, the supernatants were removed and centrifuged. The pellet obtained from 60 ml of heparinized blood was taken up in 2 ml of distilled water and alternately frozen and thawed 10 times using an ice-salt bath. The mixture was then ground in a small glass homogenizer and centrifuged. The clear pink supernatant was brought to a volume of 3 ml.Synovial membrane extracts. Synovial membrane was obtained through the cooperation of Dr. R. L. Preston from patients with rheumatoid arthritis, ankylosing spondylitis, and osteoarthritis of the hip and knee, who were undergoing joint surgery. The fresh synovial tissue was minced with scissors in the cold. Approximately 2.5 g of minced tissue was homogenized in 2.5 ml of water using a glass homogenizer. The homogenate was then alternately frozen and thawed 10 times using an ice-salt mixture and cleared by centrifugation.Viscosity measurements. Four ml of a 0.5 per cent solution of the chondromucoprotein in neutral salinephosphate buffer (0.075 M sodium chloride and 0.075 M phosphate) was pipetted into a 4 ml Ostwald viscosimeter placed in a water bath at 37°C. Following a period of 15 minutes for tem...

Immunopathologic Significance of Cartilage Antigenic Components in Rheumatoid Arthritis

“…An analogous situation may exist in rheumatoid arthritis and other inflammatory arthritides as a consequence of proteolytic enzyme depletion of matrix. In vitro and in vivo studies have shown the capacity of lysosomal enzymes to degrade the PPC of cartilage matrix (28)(29)(30). Collagenase synthesized by rheumatoid synoviocytes in in vitro culture experiments has been shown capable of producing chondrolysis (31).…”

On papain and flop–eared rabbits

“…22 With another in vitro system, it was found that bovine plasmin had the property of liberating chondroitin sulfate from slices of cartilage. 23 In view of the apparent complexity of the factors that determine the effects of papain in vivo, it is clear that the properties of mammalian enzymes in intact animals cannot be predicted with certainty from the changes produced in CMP or in isolated cartilage.…”

Degradation of cartilage proteoglycan by human leukocyte granule neutral proteases--a model of joint injury. I. Penetration of enzyme into rabbit articular cartilage and release of 35SO4-labeled material from the tissue.

“…The pH of synovial fluid in various joint diseases is generally near neutrality (6.6-7.4) (6). Furthermore, crude extracts of human PMN cytoplasmic granules (G) and neutral protease-enriched fractions isolated from these granules can degrade cartilage matrix proteoglycans in tissue slices at neutral pH (7)(8)(9)(10). More recently, at least three neutral proteases have been identified and purified from human PMN: a collagenase, an elastase (E), and a chymotrypsin-like enzyme (CT).…”