Release of Cartilage Mucopolysaccharide-Degrading Neutral Protease From Human Leukocytes

Abstract

The granule fraction of human leukocytes contains neutral protease capable of degrading the noncollagenous protein mucopolysaccharide matrix of cartilage at neutral pH in physiological salt solution. Cartilage degradation was monitored by quantitating the release of 35S from labeled rabbit ear cartilage. Degradation of cartilage matrix occurs when intact viable human leukocytes are incubated with cartilage opsonized with aggregated human gamma globulin (AHGG). During a similar 4 h incubation period cells did not degrade uncoated cartilage or cartilage coated with nonaggregated gamma globulin. Cells remain viable during the enzyme release process as evidenced by the absence of a cytoplasmic enzyme marker (lactic dehydrogenase) in the supernatant and dye exclusion studies. The release of 35S from labeled cartilage by human leukocytes in the presence of cartilage coated with AHGG (nonphagocytic enzyme release) was compared with the cartilage degrading activity of the supernatant from the same number of cells preincubated with a suspension of AHGG (phagocytic enzyme release). Nonphagocytic enzyme release by 5 x 106 cells provoked two to four times more 35S and ß-glucuronidase (ß-G) release from cartilage than phagocytic enzyme release conditions. ß-glucuronidase was used as an indicator of the release of lysosomal granule enzymes. By the use of selected pharmacological agents it was possible to dissociate the enzyme release process from intrinsic enzyme (neutral protease) activity. Neutral protease and ß-G release by human cells in the presence of AHGG-coated cartilage was inhibited by 10–5M colchicine, whereas the protease activity, but not the release process, was inhibited by 10–6M gold thiomalate and 10% human serum. It is suggested that the release of a cartilage degrading neutral protease by viable human cells when exposed to AHGG might be a relevant model for the study of cartilage destruction as it occurs in rheumatoid arthritis.

Identification of neutral proteases in human neutrophil granules that degrade articular cartilage proteoglycan

“…Instead, PMN granules alone were used in this study. On the other hand, results similar to those obtained here with isolated PMN granules have also been reported by investigators employing viable PMN incubated with immune complexes in proximity to the cartilage substrate (4,39). In such experiments with whole, living cells.…”

“…One is the absence from the described test system, and similar in vitro systems used by others (2,4,5), of serum antiproteases present in inflammatory synovial effusions (19,34). These substances can counteract the neutrophil proteases and to some degree reduce the resultant tissue injury.…”

EFFECT OF INDOMETHACIN AND RELATED DRUGS ON THE CALCIUM ION‐DEPENDENT SECRETION OF LYSOSOMAL AND OTHER ENZYMES BY NEUTROPHIL POLYMORPHONUCLEAR LEUCOCYTES In vitro

“…This is made more probable by the fact that phenylacetate and gentisate, non-antiinflammatory analogues of indomethacin and salicylate respectively (Northover, 1963;1964;Durant, Smith, Spickett & Szarvasi, 1965) (Rajan, 1966;Andrews & Phelps, 1971;Oronsky, Ignarro & Perper, 1973;Weissmann, 1973;Wright & Malawista, 1973;Hawkins, 1974;Ignarro, 1974a;Perper & Oronsky, 1974;Ackerman & Beebe, 1975;Goldstein, 1975;Lewis & Day, 1975;Malawista, 1975;Oronsky & Perper, 1975). As noted previously, stimulus-secretion coupling mechanisms may vary with the nature of the stimulus involved, and consequently may vary in their capability of being inhibited by drugs.…”

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).…”

“…In the accompanying paper, the effect of these same enzymes on isolated proteoglycans derived from cartilage is described. For the studies with whole tissue, we employed the technique of monitoring 'SO4 release from prelabeled rabbit articular cartilage, as this method has been used by others (9,10) in similar studies. The vast bulk of administered 'SO4 that enters hyaline cartilage becomes incorporated into the proteoglycans thereof (12,13).…”