Elastase and cathepsin G of human monocytes. Quantification of cellular content, release in response to stimuli, and heterogeneity in elastase-mediated proteolytic activity

Human peripheral blood monocytes contain human leukocyte elastase (HLE) and cathepsin G (CG), serine proteinases originally described in azurophil granules of polymorphonuclear neutrophils (PMN). Immunoreactive HLE and CG of freshly harvested monocytes have been quantified in this study; to begin to elucidate potential roles for these enzymes in extracellular events, release in response to stimuli has been measured, along with proteolytic activity of monocytes toward surface-bound proteins. Our results indicate that whole-cell extracts of monocytes contain approximately 6% of the amount of HLE as do extracts of comparable numbers of PMN. In response to PMA in vitro, monocytes released 39 to 53% of their content of HLE and CG within 60 min, a fractional release greater than that of PMN. Furthermore, when phorbol-stimulated monocytes were adherent to a fibronectin-coated surface, extensive HLE-mediated proteolysis of the surface-bound protein was observed. Proteolysis by such cells in the presence of proteinase inhibitors was of considerable interest, since a subpopulation (15 to 20% of the total) expressed marked but localized proteolytic activity, possibly escaping inhibition through contact-mediated mechanisms. These data indicate that a subpopulation of freshly harvested monocytes is rich in HLE and CG (serine proteinases traditionally associated with PMN), can promptly release HLE and CG in response to stimuli, and can utilize HLE for extracellular proteolysis. Monocyte-derived serine proteinases may participate in extracellular events formerly associated with PMN-derived HLE and CG.     

Leukocyte cell surface proteinases: regulation of expression, functions, and mechanisms of surface localization

A number of proteinases are expressed on the surface of leukocytes including members of the serine, metallo-, and cysteine proteinase superfamilies. Some proteinases are anchored to the plasma membrane of leukocytes by a transmembrane domain or a glycosyl phosphatidyl inositol (GPI) anchor. Other proteinases bind with high affinity to classical receptors, or with lower affinity to integrins, proteoglycans, or other leukocyte surface molecules. Leukocyte surface levels of proteinases are regulated by: (1) cytokines, chemokines, bacterial products, and growth factors which stimulate synthesis and/or release of proteinases by cells; (2) the availability of surface binding sites for proteinases; and/or (3) internalization or shedding of surface-bound proteinases. The binding of proteinases to leukocyte surfaces serves many functions including: (1) concentrating the activity of proteinases to the immediate pericellular environment; (2) facilitating pro-enzyme activation; (3) increasing proteinase stability and retention in the extracellular space; (4) regulating leukocyte function by proteinases signaling through cell surface binding sites or other surface proteins; and (5) protecting proteinases from inhibition by extracellular proteinase inhibitors. There is strong evidence that membrane-associated proteinases on leukocytes play critical roles in wound healing, inflammation, extracellular matrix remodeling, fibrinolysis, and coagulation. This review will outline the biology of membrane-associated proteinases expressed by leukocytes and their roles in physiologic and pathologic processes.     

An unusual specificity in the activation of neutrophil serine proteinase zymogens

The majority of proteinases exist as zymogens whose activation usually results from a single proteolytic event. Two notable exceptions to this generalization are the serine proteinases neutrophil elastase (HNE) and cathepsin G (cat G), proteolytic enzymes of human neutrophils that are apparently fully active in their storage granules. On the basis of amino acid sequences inferred from the gene and cDNAs encoding these enzymes, it is likely that both are synthesized as precursors containing unusual C-terminal and N-terminal peptide extensions absent from the mature proteins. We have used biosynthetic radiolabeling and radiosequencing techniques to identify the kinetics of activation of both proteinases in the promonocyte-like cell line U937. We find that both N- and C-terminal extensions are removed about 90 min after the onset of synthesis, resulting in the activation of the proteinases. HNE and cat G are, therefore, transiently present as zymogens, presumably to protect the biosynthetic machinery of the cell from adventitious proteolysis. Activation results from cleavage following a glutamic acid residue to give an activation specificity opposite to those of almost all other serine proteinase zymogens, but shared, possibly, by the "granzyme" group of related serine proteinases present in the killer granules of cytotoxic T-lymphocytes and rat mast cell proteinase II.     

Neutrophil serine proteinases cleave bacterial flagellin, abrogating its host response-inducing activity

After bacterial infection, neutrophils dominate the cellular infiltrate. Their main function is assumed to be killing invading pathogens and resolving the inflammation they cause. Activated neutrophils are also known to release a variety of molecules, including the neutrophil serine proteinases, extracellularly. The release of these proteinases during inflammation creates a proteolytic environment where degradation of different molecules modulates the inflammatory response. Flagellin, the structural component of flagella on many bacterial species, is a virulence factor with a strong proinflammatory activity on epithelial cells and other cell types. In this study we show that both human and mouse neutrophil serine proteinases cleave flagellin from Pseudomonas aeruginosa and other bacterial species. More important, cleavage of P. aeruginosa flagellin by the neutrophil serine proteinases neutrophil elastase and cathepsin G resulted in loss of the biological activity of this virulence factor, as evidenced by the lack of innate host defense gene expression in human epithelial cells. The finding that flagellin is susceptible to cleavage by neutrophil serine proteinases suggests a novel role for these enzymes in the inflammatory response to infection. Not only can these enzymes kill bacteria, but they also degrade their virulence factors to halt the inflammatory response they trigger.     

Novel roles of protease inhibitors in infection and inflammation

The local balance between proteinase inhibitors and proteinases determines local proteolytic activity. Various studies have demonstrated the importance of serine proteinase inhibitors in regulating the activity of serine proteinases that are released by leucocytes during inflammation. Recently it has been shown that these inhibitors may also display functions that are distinct from those associated with the inhibition of leucocyte-derived proteinases. In this review the results of selected studies focusing on three inhibitors of neutrophil elastase, i.e. alpha(1)-proteinase inhibitor, secretory leucocyte proteinase inhibitor and elafin, are presented, with the aim of illustrating their possible involvement in the regulation of inflammation, host defence against infection, tissue repair and extracellular matrix synthesis.     

Elastase and cathepsin G of human monocytes: heterogeneity and subcellular localization to peroxidase-positive granules

We used antibodies to human leukocyte ("neutrophil") elastase and cathepsin G to localize the corresponding antigens in human neutrophils, monocytes, and alveolar macrophages by immunohistochemistry. Furthermore, we combined immunogold localization with enzyme histochemistry to localize proteinase antigens and endogenous peroxidase activity in the same sections. As expected, all neutrophils contained both elastase and cathepsin G, and the proteinases localized to granules with peroxidase activity. In contrast, marked heterogeneity in monocyte staining for elastase, cathepsin G, and endogenous peroxidase was found. Sixty percent or more were unstained, while the remainder varied greatly in staining intensity. The elastase and cathepsin G in monocytes were localized by immunoelectron microscopy, combined with histochemistry, to cytoplasmic granules which had peroxidase activity. Alveolar macrophages were unstained. Therefore, a subpopulation of peripheral blood monocytes contains leukocyte elastase and cathepsin G in a cell compartment from which these enzymes may potentially be released into the extracellular space. The occurrence of peroxidase and neutral proteinases in the same granules in monocytes could permit the H2O2-myeloperoxidase-halide system and the neutral proteinases to act in concert in such functions as microbe killing and extracellular proteolysis.     

Identification of a cathepsin G-like proteinase in the MCTC type of human mast cell

Human mast cells can be divided into two subsets based on serine proteinase composition: a subset that contains the serine proteinases tryptase and chymase (MCTC), and a subset that contains only tryptase (MCT). In this study we examined both types of mast cells for two additional proteinases, cathepsin G and elastase, which are the major serine proteinases of neutrophils. Because human mast cell chymase and cathepsin G are both chymotrypsin-like proteinases, the properties of these enzymes were further defined to confirm their distinctiveness. Comparison of their N-terminal sequences showed 30% nonidentity over the first 35 amino acids, and comparison of their amino acid compositions demonstrated a marked difference in their Arg/Lys ratios, which was approximately 1 for chymase and 10 for cathepsin G. Endoglycosidase F treatment increased the electrophoretic mobility of chymase on SDS gels, indicating significant N-linked carbohydrate on chymase; no effect was observed on cathepsin G. Immunoprecipitation and immunoblotting with specific antisera to each proteinase revealed little, if any, detectable cross-reactivity. Immunocytochemical studies showed selective labelling of MCTC type mast cells by cathepsin G antiserum in sections of human skin, lung, and bowel. No labeling of mast cells by elastase antiserum was detected in the same tissues, or in dispersed mast cells from lung and skin. A protein cross-reactive with cathepsin G was identified in extracts of human skin mast cells by immunoblot analysis. This protein had a slightly higher Mr (30,000) than the predominant form of neutrophil cathepsin G (Mr 28,000), and could not be separated from chymase (Mr 30,000) by SDS gel electrophoresis because of the size similarity. Using casein, a protein substrate hydrolyzed at comparable rates by chymase and cathepsin G, it was shown that about 30% of the caseinolytic activity in mast cell extracts was sensitive to inhibitors of cathepsin G that had no effect on chymase. Hydrolytic activity characteristic of elastase was not detected in these extracts. These studies indicate that human MCTC mast cells may contain two different chymotrypsin-like proteinases: chymase and a proteinase more closely related to cathepsin G, both of which are undetectable in MCT mast cells. Neutrophil elastase, on the other hand, was not detected in human mast cells by our procedures.     

Different susceptibility of elastase inhibitors to inactivation by proteinases from Staphylococcus aureus and Pseudomonas aeruginosa.

Neutrophil elastase is thought to contribute to the lung pathology in patients with cystic fibrosis (CF). Therefore, intrapulmonary application of elastase inhibitors might be beneficial for these patients. Inactivation of such inhibitors by bacterial proteinases, however, is an important consideration in this therapy. We studied the effects of Staphylococcus aureus proteinase (STAP) and Pseudomonas aeruginosa elastase (PsE) on native (alpha 1-AT) and recombinant (rAAT) alpha 1-antitrypsin, recombinant secretory leukocyte proteinase inhibitor (rSLPI) and the leech inhibitor eglin C. All inhibitors were inactivated by these bacterial proteinases showing pronounced differences in their susceptibilities to proteolytic cleavage. Comparing the turnover rate (mol of inhibitor inactivated by one mol bacterial proteinase/min), rAAT and alpha 1-AT were approximately 20,000-fold more susceptible to STAP than rSLPI and 50,000-fold more susceptible than eglin C. Pseudomonas aeruginosa elastase inactivated all inhibitors more rapidly than STAP. rAAT and alpha 1-AT were 13-fold and 17,000-fold more susceptible than rSLPI and eglin C, respectively. Incubation of the rAAT-elastase complex with equimolar amounts of STAP did not result in release of elastase activity. Upon simultaneous addition of STAP and leukocyte elastase to rAAT, there was undisturbed elastase inhibition indicating that complex formation with elastase proceeded at a faster rate than inactivation of rAAT by the bacterial proteinase. From these results of inactivation in vitro and considering the immunogenic potential of the inhibitors studied here, we conclude that rSLPI may be the appropriate choice for anti-elastase therapy in CF.     

Bioactive proteinase 3 on the cell surface of human neutrophils: quantification, catalytic activity, and susceptibility to inhibition

Although proteinase 3 (PR3) is known to have the potential to promote inflammation and injure tissues, the biologic forms and function of PR3 in polymorphonuclear neutrophils (PMN) from healthy donors have received little attention. In this paper, we show that PMN contain 3.24 +/- SD 0.24 pg of PR3 per cell, and that the mean concentration of PR3 in azurophil granules of PMN is 13.4 mM. Low levels of PR3 are detectable on the cell surface of unstimulated PMN. Exposure of PMN to cytokines or chemoattractants alone induces modest (1.5- to 2.5-fold) increases in cell surface-bound PR3. In contrast, brief priming of PMN with cytokines, followed by activation with a chemoattractant, induces rapid and persistent, 5- to 6-fold increases in cell surface expression of PR3, while causing minimal free release of PR3. Membrane-bound PR3 on PMN is catalytically active against Boc-Alanine-Alanine-Norvaline-thiobenzyl ester and fibronectin, but in marked contrast to soluble PR3, membrane-bound PR3 is resistant to inhibition by physiologic proteinase inhibitors. PR3 appears to bind to the cell surface of PMN via a charge-dependent mechanism because exposure of fixed, activated PMN to solutions having increasing ionic strength results in elution of PR3, HLE, and CG, and there is a direct relationship between their order of elution and their isoelectric points. These data indicate that rapidly inducible PR3 expressed on the cell surface of PMN is an important bioactive form of the proteinase. If PR3 expression on the cell surface of PMN is dysregulated, it is well equipped to amplify tissue injury directly, and also indirectly via the generation of autoantibodies.     

Inhibition of neutrophil elastase by alpha1-protease inhibitor at the surface of human polymorphonuclear neutrophils

The uncontrolled proteolytic activity in lung secretions during lung inflammatory diseases might be due to the resistance of membrane-bound proteases to inhibition. We have used a new fluorogenic neutrophil elastase substrate to measure the activity of free and membrane-bound human neutrophil elastase (HNE) in the presence of alpha1-protease inhibitor (alpha1-Pi), the main physiological inhibitor of neutrophil serine proteases in lung secretions. Fixed and unfixed neutrophils bore the same amounts of active HNE at their surface. However, the HNE bound to the surface of unfixed neutrophils was fully inhibited by stoichiometric amounts of alpha1-Pi, unlike that of fixed neutrophils. The rate of inhibition of HNE bound to the surface of unfixed neutrophils was the same as that of free HNE. In the presence of alpha1-Pi, membrane-bound elastase is almost entirely removed from the unfixed neutrophil membrane to form soluble irreversible complexes. This was confirmed by flow cytometry using an anti-HNE mAb. HNE activity rapidly reappeared at the surface of HNE-depleted cells when they were triggered with the calcium ionophore A23187, and this activity was fully inhibited by stoichiometric amounts of alpha1-Pi. HNE was not released from the cell surface by oxidized, inactive alpha1-Pi, showing that active inhibitor is required to interact with active protease from the cell surface. We conclude that HNE activity at the surface of human neutrophils is fully controlled by alpha1-Pi when the cells are in suspension. Pericellular proteolysis could be limited to zones of contact between neutrophils and subjacent protease substrates where natural inhibitors cannot penetrate.     

Apoptotic neutrophils undergoing secondary necrosis induce human lung epithelial cell detachment

Clearance of apoptotic neutrophils by alveolar macrophages plays an important role in the resolution phase of lung inflammation. If not cleared, apoptotic neutrophils are postulated to release histotoxic granular contents. Since numerous cellular proteins are degraded during apoptosis, we sought to determine whether functional serine proteinases are indeed released by apoptosing neutrophils in vitro. In a coculture system, cytokine-activated neutrophils induced detachment in the human epithelial cell line, A549. This process was CD18- and serine proteinase-dependent. Early apoptotic neutrophils induced significant detachment, but live, senescent, resting neutrophils and terminal, secondary necrotic neutrophils had a different effect. This detachment process was CD18-independent but serine proteinase-dependent. Similarly, detachment occurred with primary human small airway epithelial cells. Notably, epithelial cell detachment correlated with the transition of early apoptotic neutrophils to secondary necrosis and with the accumulation of elastase in the supernatant. The membrane integrity of lung epithelial cells was damaged in advance of significant cell detachment. These observations suggest that not only live activated neutrophils but also apoptosing neutrophils can reveal functional elastase activities. Furthermore, the rapidity of the transition emphasizes the importance of the prompt clearance of apoptotic neutrophils before they progress to secondary necrosis at the site of lung inflammation.C.Y.L. and Y.H.L contributed equally to the work on this project as first authors.     

Neutrophil serine proteinases inactivate surfactant protein D by cleaving within a conserved subregion of the carbohydrate recognition domain

Surfactant protein D (SP-D) plays important roles in innate immunity including the defense against bacteria, fungi, and respiratory viruses. Because SP-D specifically interacts with neutrophils that infiltrate the lung in response to acute inflammation and infection, we examined the hypothesis that the neutrophil-derived serine proteinases (NSPs): neutrophil elastase, proteinase-3, and cathepsin G degrade SP-D. All three human NSPs specifically cleaved recombinant rat and natural human SP-D dodecamers in a time- and dose-dependent manner, which was reciprocally dependent on calcium concentration. The NSPs generated similar, relatively stable, disulfide cross-linked immunoreactive fragments of approximately 35 kDa (reduced), and sequencing of a major catheptic fragment definitively localized the major sites of cleavage to a highly conserved subregion of the carbohydrate recognition domain. Cleavage markedly reduced the ability of SP-D to promote bacterial aggregation and to bind to yeast mannan in vitro. Incubation of SP-D with isolated murine neutrophils led to the generation of similar fragments, and cleavage was inhibited with synthetic and natural serine proteinase inhibitors. In addition, neutrophils genetically deficient in neutrophil elastase and/or cathepsin G were impaired in their ability to degrade SP-D. Using a mouse model of acute bacterial pneumonia, we observed the accumulation of SP-D at sites of neutrophil infiltration coinciding with the appearance of approximately 35-kDa SP-D fragments in bronchoalveolar lavage fluids. Together, our data suggest that neutrophil-derived serine proteinases cleave SP-D at sites of inflammation with potential deleterious effects on its biological functions.     

Neutrophil elastase, proteinase 3 and cathepsin G: physicochemical properties, activity and physiopathological functions

Polymorphonuclear neutrophils form a primary line of defense against bacterial infections using complementary oxidative and non-oxidative pathways to destroy phagocytized pathogens. The three serine proteases elastase, proteinase 3 and cathepsin G, are major components of the neutrophil primary granules that participate in the non-oxidative pathway of intracellular pathogen destruction. Neutrophil activation and degranulation results in the release of these proteases into the extracellular medium as proteolytically active enzymes, part of them remaining exposed at the cell surface. Extracellular neutrophil serine proteases also help kill bacteria and are involved in the degradation of extracellular matrix components during acute and chronic inflammation. But they are also important as specific regulators of the immune response, controlling cellular signaling through the processing of chemokines, modulating the cytokine network, and activating specific cell surface receptors. Neutrophil serine proteases are also involved in the pathogenicity of a variety of human diseases. This review focuses on the structural and functional properties of these proteases that may explain their specific biological roles, and facilitate their use as molecular targets for new therapeutic strategies.     

Leukocyte chemoattractant peptides from the serpin heparin cofactor II

Heparin cofactor II (HC) is a plasma serine proteinase inhibitor (serpin) that inhibits the coagulant proteinase alpha-thrombin. We have recently demonstrated that proteolysis of HC by catalytic amounts of polymorphonuclear leukocyte proteinases (elastase or cathepsin G) generates leukocyte chemotaxins (Hoffman, M., Pratt, C. W., Brown, R. L., and Church, F. C. (1989) Blood 73, 1682-1685). One of four peptides produced when HC is degraded by neutrophil elastase has chemotactic activity for both monocytes and neutrophils with maximal migration comparable to formyl-Met-Leu-Phe, the "gold standard" bacterially derived chemotaxin. The amino-terminal sequence of this HC peptide is Asp-Phe-His-Lys-Glu-Asn-Thr-Val-... and the peptide corresponds to Asp-39 to Ile-66 of HC. A variety of synthetic peptides derived from this sequence were evaluated for leukocyte migration activity, and a dodecapeptide from Asp-49 to Tyr-60 (Asp-Trp-Ile-Pro-Glu-Gly-Glu-Glu-Asp-Asp-Asp-Tyr) was identified as the active site for leukocyte chemotactic action. The 12-mer synthetic peptide possesses significant neutrophil chemotactic action at 1 nM (60% of the maximal activity of formyl-Met-Leu-Phe), while a peptide with the reverse sequence has essentially no chemotactic activity. Cross-desensitization experiments also show that pretreatment of neutrophils with a 19-mer peptide (Asn-48 to Ile-66) greatly reduces subsequent chemotaxis to HC-neutrophil elastase proteolysis reaction products. When injected intraperitoneally in mice, the HC-neutrophil elastase digest elicits neutrophil migration. Our results demonstrate that not only does HC function as a thrombin inhibitor, but that limited proteolysis of HC near the amino terminus yields biologically active peptide(s) which might participate in inflammation and in wound healing and tissue repair processes.     

Characterisation and properties of ectosomes released by human polymorphonuclear neutrophils

Human neutrophils release vesicles when activated in vitro and in vivo, in local and systemic inflammation. We have suggested that the presence of these vesicles is due to ectocytosis, defined as the release of rightside-out oriented vesicles expressing a select set of membrane proteins. Herein we have characterised the vesicles released by neutrophils to be ectosomes with specific properties. They contained cytosolic F-actin indicating their outside-out orientation. They bound Annexin V, suggesting that they expose phosphatidylserine, similarly to platelet microparticles. They expressed a subset of cell surface proteins (selectins and integrins, complement regulators, HLA-1, FcgammaRIII, and CD66b, but not CD14, FcgammaRII, and CD87). There was no specificity for transmembrane or glycosyl-phosphatidylinositol-linked proteins and, unexpectedly, L-selectin, known to be cleaved from the surface of activated neutrophils, was present. Ectosomes exposed active enzymes released by neutrophils upon degranulation (matrix metalloproteinase-9, myeloperoxidase, proteinase 3, and elastase). In particular, released myeloperoxidase was able to bind back to ectosomes. The purified complement protein C1q and C1q from serum bound to ectosomes as well. Another aspect of ectosomes was that they became specifically adherent to monocytic and endothelial cells. These observations suggest that neutrophil-derived ectosomes have unique characteristics that make them candidates for playing roles in inflammation and cell signaling.     

The sulfate groups of chondroitin sulfate- and heparan sulfate-containing proteoglycans in neutrophil plasma membranes are novel binding sites for human leukocyte elastase and cathepsin G

Human leukocyte elastase (HLE) and cathepsin G (CG) are expressed at high levels on the surface of activated human neutrophils (PMN) in catalytically active but inhibitor-resistant forms having the potential to contribute to tissue injury. Herein we have investigated the mechanisms by which HLE and CG bind to PMN plasma membranes. (125)I-Labeled HLE and CG bind to PMN at 0 degrees C in a saturable and reversible manner (K(D) = 5.38 and 4.36 x 10(-7) m and 11.5 and 8.1 x 10(6) binding sites/cell, respectively). Incubation of PMN with radiolabeled HLE and CG in the presence of a 200-fold molar excess of unlabeled HLE, CG, myeloperoxidase, lactoferrin, proteinase 3, phenylmethylsulfonyl fluoride (PMSF)-inactivated HLE, or PMSF-inactivated CG inhibited binding of radiolabeled ligands. This indicates that these PMN granule proteins share binding sites on PMN and that functional active sites of HLE and CG are not required for their binding to PMN. The sulfate groups of heparan sulfate- and chondroitin sulfate-containing proteoglycans are the PMN binding sites for HLE and CG since binding of HLE and CG to PMN was inhibited by incubating PMN with 1) trypsin, chondroitinase ABC, and heparitinases, but not other glycanases, and 2) purified chondroitin sulfates, heparan sulfate, and other sulfated molecules, but not with non-sulfated glycans. Thus, heparan sulfate- and chondroitin sulfate-containing proteoglycans are low affinity, high volume PMN surface binding sites for HLE and CG, which are well suited to bind high concentrations of active serine proteinases released from degranulating PMN.     

Proteinase inhibitors in rat serum. Purification and partial characterization of three functionally distinct trypsin inhibitors.

Three different serine proteinase inhibitors were isolated from rat serum and purified to apparent homogeneity. One of the inhibitors appears to be homologous to alpha 1-proteinase inhibitor isolated from man and other species, but the other two, designated rat proteinase inhibitor I and rat proteinase inhibitor II, seem to have no human counterpart. alpha 1-Proteinase inhibitor (Mr 55000) inhibits trypsin, chymotrypsin and elastase, the three serine proteinases tested. Rat proteinase inhibitor I (Mr 66000) is active towards trypsin and chymotrypsin, but is inactive towards elastase. Rat proteinase inhibitor II (Mr 65000) is an effective inhibitor of trypsin only. Their contributions to the trypsin-inhibitory capacity of rat serum are about 68, 14 and 18% for alpha 1-proteinase inhibitor, rat proteinase inhibitor I and rat proteinase inhibitor II respectively.     

Proteinase inhibitory spectrum of mouse murinoglobulin and alpha-macroglobulin

The interactions of mouse murinoglobulin and alpha-macroglobulin with several proteinases were investigated by filtration and by assays of amidolytic activity towards synthetic substrates in the presence of proteinaceous enzyme inhibitors as well as assays of the inhibition of proteolytic activity. Mouse alpha-macroglobulin formed complexes with thrombin, clotting factor Xa, plasmin, pancreatic kallikrein, plasma kallikrein, submaxillary gland trypsin-like proteinase, neutrophil elastase, and pancreatic elastase. These complexes lost the proteolytic activities against high-molecular-weight substrates, but protected the active sites of the enzymes from inactivation by their proteinaceous inhibitors. Mouse murinoglobulin showed essentially the same properties except (i) that it did not form a complex with the clotting factor Xa, and (ii) that it did not protect plasma kallikrein, neutrophil elastase or submaxillary proteinase from inactivation by their proteinaceous inhibitors, although it formed complexes with these proteinases. No interaction was detected between Clostridium histolyticum collagenase and murinoglobulin or alpha-macroglobulin. These results indicate (i) that murinoglobulin has a proteinase-binding spectrum similar to that of alpha-macroglobulin, but is weaker in the ability to protect the bound proteinases from inactivation by the proteinaceous inhibitors than alpha-macroglobulin and (ii) that mouse alpha-macroglobulin has essentially the same inhibitory spectrum as the human homologue.     

The effect of oxidants on neutrophil-mediated degradation of glomerular basement membrane collagen

The contribution of activated oxygen species to neutrophil-mediated degradation of basement membrane collagen was investigated. In preliminary experiments, pre-exposure of either albumin or glomerular basement membrane to neutrophil myeloperoxidase with H2O2 and chloride increased their susceptibility to proteolysis 2-3-fold. In the basement membrane model, neutrophils are stimulated by trapped immune complexes to adhere, produce oxidants and degranulate. Degradation, measured as the amount of hydroxyproline solubilised, was due to neutral proteinases, particularly elastase, and depended on cell number and the amount of proteinase released. Experiments with oxidant scavengers and inhibitors and with neutrophils from donors with chronic granulomatous disease or myeloperoxidase deficiency showed that oxidants did not affect degradation of the basement membrane when this was measured on a per cell basis. However, oxidative inactivation of the released granule enzymes occurred. Activities of elastase, beta-glucuronidase and lysozyme were 1.5-2-times higher in the presence of catalase, but were unaffected by superoxide dismutase or hydroxyl radical scavengers. Inactivation did not occur with chronic granulomatous disease or myeloperoxidase deficient neutrophils. When related to the activity of released elastase, or to other degranulation markers, collagen degradation was decreased in the presence of catalase, or with chronic granulomatous disease or myeloperoxidase deficient cells. This implies that the basement membrane was made more digestible by myeloperoxidase-derived oxidants, as occurred in the cell-free experiments. Taken together, the results indicate that neutrophil oxidants have two opposing effects. They increase the susceptibility of the collagen to proteolysis and inactivate the proteinases responsible.     

Proteinases involved in matrix turnover during cartilage and bone breakdown

The joint is a discrete unit that consists of cartilage, bone, tendon and ligaments. These tissues are all composed of an extracellular matrix made of collagens, proteoglycans and specialised glycoproteins that are actively synthesised, precisely assembled and subsequently degraded by the resident connective tissue cells. A balance is maintained between matrix synthesis and degradation in healthy adult tissues. Different classes of proteinases play a part in connective tissue turnover in which active proteinases can cleave matrix protein during resorption, although the proteinase that predominates varies between different tissues and diseases. The metalloproteinases are potent enzymes that, once activated, degrade connective tissue and are inhibited by tissue inhibitors of metalloproteinases (TIMPs); the balance between active matrix metalloproteinases and TIMPs determines, in many tissues, the extent of extracellular matrix degradation. The serine proteinases are involved in the initiation of activation cascades and some, such as elastase, can directly degrade the matrix. Cysteine proteinases are responsible for the breakdown of collagen in bone following the removal of the osteoid layer and the attachment of osteoclasts to the exposed bone surface. Various growth factors increase the synthesis of matrix and proteinase inhibitors, whereas cytokines (alone or in combination) can inhibit matrix synthesis and stimulate proteinase production and matrix destruction.