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

Abstract

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.

A critical role for neutrophil elastase in experimental bullous pemphigoid

“…Consistent with this hypothesis are the recent findings that mutations within the BP180 gene lead to the inherited blistering disease, generalized atrophic benign epidermolysis bullosa (49). NE degrades collagens (50-53), fibronectin (54), and proteoglycans (55,56). The results of our in vitro enzyme digestion experiments demonstrate that NE also cleaves the extracellular domain of the BP180 protein.…”

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 mol wt of the principal CT isozyme lies between 23,000 and 28,000 daltons, and its amino acid composition has now been described with enzyme derived from both leukemic (35) and normal (18) cells. In an earlier report from this laboratory (15) it was suggested that these two PMN enzymes, E and CT, were the major proteases responsible for degradation of cartilage matrix proteoglycans by human PMN granule extracts (G) at neutral pH. This tentative conclusion was based on the observed solubilization of 'SO4 from slices of rabbit articular cartilage by partly purified protein fractions isolated by preparative acrylamide gel electrophoresis 620 A. Janoff, G. Feinstein, C. J.…”

“…This tentative conclusion was based on the observed solubilization of 'SO4 from slices of rabbit articular cartilage by partly purified protein fractions isolated by preparative acrylamide gel electrophoresis 620 A. Janoff, G. Feinstein, C. J. Malemud of G. Only those acrylamide gel fractions containing E or CT were active in releasing the radiolabel (15).…”

“…The proximal humeri and tibiae and the proximal and distal femora were removed and slices of articular cartilage were then dissected from the joints in the cold and subsequently stored at -800C. Further details of these procedures were described earlier (15).…”

Inhibition of neutrophil elastase by the α1‐proteinase inhibitor‐immunoglobulin A complex

“…Massive numbers of neutrophils accumulate in rheumatoid synovial fluids where they may release large amounts of NE [1], a serine proteinase that cleaves proteoglycans and type II collagen, the major constituents of articular cartilage [2,3]. NE is therefore believed to play a significant pathological role in arthritis.…”