Breakdown of noncollagenous chondromucoprotein matrix by leukocyte lysosome granule lysates from guinea pig, rabbit, and human

Ignarro, Louis J.; Oronsky, Arnold L.; Perper, R. J.

doi:10.1016/0090-1229(73)90034-2

Cited by 55 publications

(30 citation statements)

References 32 publications

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“…These two esterases would be expected to be present in zone 3 of our electre phoretograms. Moreover, Ignarro et al (5) found that pancreatic chymotrypsin degrades rabbit ear cartilage proteoglycan. In the present experiments, it was found that chymotrypsin (1 Lcg/ml) released 71% of the available radiosulfate from rabbit articular cartilage proteoglycan in ?L2 hour.…”

Section: Resultsmentioning

confidence: 99%

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

Malemud

Janoff

1975

Arthritis & Rheumatism

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Human polymorphonuclear neutrophil (PMN) granule extract (25 pg of protein) released 60% of the available 3 5 s 0 , from labeled rabbit articular cartilage in 0.5 hour at neutral pH. N-acetyl-L-alanyl-Lalanyl-L-prolyl-L-alanine chloromethyl ketone (NAc-AAPACK), a specific elastase inhibitor, was only minimally effective against whole granule extract, and Na-tosyl-L-lysine chloromethyl ketone, which inhibits trypsin but not elastase, was completely ineffective. Preparative disc-gel electrophoresis of PMN granule extract revealed two separate regions with independent activity against 35SO,-labeled cartilage. One region contained elastases and, when tested alone, was completely inhibited by NAcAAPACK. The other contained lysozyme and two esterases active against Nacetyl-L-phenylalanine-a-naphthol. proved inactive, suggesting that the chymotrypsin-like esterases were responsible for proteoglycan degradation by this region of the gel.The final common pathway hypothesis of joint diseases implies that damage to articular cartilage can be caused by lysosomal proteases in response to a number of different insults (1). Previously it was shown that human leukocytes contain lysosomal enzymes capable of degrading cartilage proteoglycan (2-5) and in a recent series of investigations (4,5) it was reported that neutral protease activity from human neutrophils can release 35S0,-labeled proteoglycan fragments from rabbit ear cartilage in vitro. However the responsible enzyme(s) was not identified. The present investigation represents a preliminary effort to achieve this identification. In the work to be reported, articular cartilage rather than ear cartilage has been used because of the following considerations: a) It is a more suitable substrate to test the potential action of neutrophil hydrolases in joint diseases, and b) it is more homogeneous than ear cartilage with respect to hyaline vis-A-vis fibrocartilaginous components. It is reported here that human polymorphonuclear (PMN) granular extracts can rapidly release 35S0, from rabbit articular cartilage proteoglycan in vitro at neutral pH and that the two major enzymes responsible for this effect resemble pancreatic elastase and a chymotrypsin-like esterase.

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Section: Resultsmentioning

confidence: 99%

“…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.…”

Section: Discussionmentioning

confidence: 99%

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

Malemud

Janoff

1975

Arthritis & Rheumatism

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“…These discs were prepared as described by Ignarro et al (1973). The cartilage cells were then killed by repeated freezing and thawing and the cartilage was washed extensively before the enzyme assays.…”

Section: Methodsmentioning

confidence: 99%

The simultaneous release by bone explants in culture and the parallel activation of procollagenase and of a latent neutral proteinase that degrades cartilage proteoglycans and denatured collagen

Vaes¹,

Eeckhout²,

Lenaers-Claeys³

et al. 1978

Biochemical Journal

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1. A latent neutral proteinase was found in culture media of mouse bone explants. Its accumulation during the cultures is closely parallel to that of procollagenase; both require the presence of heparin in the media. 2. Latent neutral proteinase was activated by several treatments of the media known to activate procollagenase, such as limited proteolysis by trypsin, chymotrypsin, plasmin or kallikrein, dialysis against 3 M-NaSCN at 4°C and prolonged preincubation at 25°C. Its activation often followed that of the procollagenase present in the same media. 3. Activation ofneutral proteinase (as does that ofprocollagenase) by trypsin or plasmin involved two successive steps: the activation of a latent endogenous activator present in the media followed by the activation of neutral proteinase itself by that activator. 4. The proteinase degrades cartilage proteoglycans, denatured collagen (Azocoll) and casein at neutral pH; it is inhibited by EDTA, cysteine or serum. Collagenase is not inhibited by casein or Azocoll and is less resistant to heat or to trypsin than is the proteinase. Partial separation of the two enzymes was achieved by gel filtration of the media but not by fractional (NH4)2SO4 precipitation, by ion exchange or by affinity chromatography on Sepharose-collagen. These fractionations did not activate latent enzymes. 5. Trypsin activation decreases the molecular weight of both latent enzymes

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“…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).…”

Section: Introductionmentioning

confidence: 99%

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.

Janoff¹,

Feinstein²,

Malemud³

et al. 1976

J. Clin. Invest.

120

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A B S T R A C T The present work was undertaken to explore the effect of two purified neutral proteases derived from human peripheral blood polymorphonuclear leukocytes (PMN) on articular cartilage as a model of joint injury. Human leukocyte elastase and chymotrypsin-like enzyme, purified by affinity chromatography, released 'SO4 from labeled rabbit articular cartilage slices in vitro. Release of isotope was initially delayed, suggesting that either a lag in enzyme penetration occurs or that size of degradation fragments is a limiting factor in diffusion of label out of the tissue. The release of 'SO4 was inhibited by preincubation of elastase and chymotrypsin-like enzyme with human alpha 1-antitrypsin, or with their specific chloromethyl ketone inactivators, and the action of elastase was also inhibited by a monospecific antiserum to PMN elastase, freed of major serum proteinase inhibitors. Immunohistochemical staining procedures revealed the presence of PMN elastase inside the matrix of cartilage slices after a 20-min exposure of tissue to either the pure enzyme or crude PMN granule extract. Serum alpha 1-antitrypsin failed to penetrate into the cartilage slices under identical in vitro conditions. In association with the results reported in the accompanying paper, these findings suggest a model of cartilage matrix degradation by PMN neutral proteases in which local protease-antiprotease imbalance, coupled with different rates of penetration of protease and antiprotease into target tissue, plays a key role in accounting for matrix damage.Dr. Feinstein's permanent address is:

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Breakdown of noncollagenous chondromucoprotein matrix by leukocyte lysosome granule lysates from guinea pig, rabbit, and human

Cited by 55 publications

References 32 publications

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

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

The simultaneous release by bone explants in culture and the parallel activation of procollagenase and of a latent neutral proteinase that degrades cartilage proteoglycans and denatured collagen

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.

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