Human Leukocyte Elastase Activity Research Articles

Biological activities of extract of Anthemis aetnensis Schouw: In vitro evaluation

Although Anthemis spp. are commonly used as anti-inflammatory, antibacterial agents, the exact mechanism of action is still unknown. We evaluated the antioxidant effect of Anthemis aetnensis by testing the free radical scavenging capacity and its effect on xanthine oxidase (XO) activity. In addition, the effect of the same extract on human leukocyte elastase activity was also evaluated. Flowers of A. aetnensis Schouw were collected on the slopes of the volcano Mount Etna. Antioxidant activity was assessed using a method that excludes the Fenton-type reaction. Since xanthine (X)/XO system represents a source of free radicals, we also tested the hypothesis that the study’s extract might inhibit the enzymatic activity of XO. In addition, the same extract of A. aetnensis was used to test its activity on human leukocyte elastase. Extract of A. aetnensis showed antioxidant activity, which was not due to inhibition of XO activity. In addition, extract of A. aetnensis showed a significant elastase inhibitory activity. Our data suggest that antioxidant capacity and inhibitor activity on elastase may explain the protective effects on inflammation, ulcers, irradiated skin injuries and cystitis afflictions. Key words: Anthemis aetnensis, reactive oxygen species, antioxidant activity, human leukocyte elastase, xanthine oxidase.

Chemically modified tetracycline-3 (CMT-3): A novel inhibitor of the serine proteinase, elastase

Two classes of enzymes play an important role in connective tissue breakdown during various inflammatory diseases: serine proteinases and matrix metalloproteinases (MMPs). Tetracyclines (TCs) exhibit important anti-inflammatory and MMP-inhibitory properties that are unrelated to their antibacterial activities. Of the various TCs and their chemically modified NON-antibiotic analogs (CMTs) tested in vitro and in vivo, CMT-3 (6-demethyl-6-deoxy 4 de-dimethylamino tetracycline) has repeatedly been shown to be the most potent inhibitor of MMP activity and cytokine production. In addition to its anti-MMP function, we have shown that among all CMTs, CMT-3 is the only CMT that can also directly inhibit both the amidolytic activity of human leukocyte elastase (HLE, a serine proteinase) and the extracellular matrix degradation mediated by HLE. In addition, CMT-3 has been found to reduce leukocyte elastase activity in vivo in gingival extracts of rats with experimental periodontal disease. Thus, CMT-3 can inhibit pathologic connective tissue breakdown by (at least) two mechanisms: direct inhibition of neutral proteinases (elastase and MMPs); and protecting their endogenous inhibitors, α1-PI and TIMPs, from being digested and inactivated by MMPs and HLE, respectively. The pleiotropic properties of CMT-3 including (but not limited to) inhibition of serine proteinases, MMPs, and cytokines provide impressive therapeutic potential to reduce excessive connective tissue breakdown during various pathologic processes including inflammatory diseases, cancer metastasis and metabolic bone diseases.

Inhibition of human leukocyte elastase, plasmin and matrix metalloproteinases by oleic acid and oleoyl-galardin derivative(s)

Molecular modeling was undertaken at aims to analyze the interactions between oleic acid and human leukocyte elastase (HLE), plasmin and matrix metalloproteinase-2 (MMP-2), involved in the inhibitory capacity of fatty acid towards those proteases. The carboxylic acid group of the fatty acid was found to form a salt bridge with Arg 217 of HLE while unsaturation interacted with Phe 192 and Val 216 at the S 3 subsite, and alkyl end group occupied S 1 subsite. In keeping with the main contribution of kringle 5 domain in plasmin–oleic acid interaction [Huet E et al. Biochem Pharmacol 2004;67(4):643–54], docking computations revealed that the long alkyl chain of fatty acid inserted within an hydrophobic groove of this domain with the carboxylate forming a salt bridge with Arg 512. Finally, blind docking revealed that oleic acid could occupy both S′ 1 subsite and Fn(II) 3 domain of MMP-2. Several residues involved in Fn(II) 3/oleic acid interaction were similarly implicated in binding of this domain to collagen. Oleic acid was covalently linked to galardin (at P′ 2 position): OL-GAL (CONHOH) or to its carboxylic acid counterpart: OL-GAL (COOH), with the idea to obtain potent MMP inhibitors able to also interfere with elastase and plasmin activity. OL-GALs were found less potent MMP inhibitors as compared to galardin and no selectivity for MMP-2 or MMP-9 could be demonstrated. Docking computations indicated that contrary to oleic acid, OL-GAL binds only to MMP-2 active site and surprisingly, hydroxamic acid was unable to chelate Zn, but instead forms a salt bridge with the N-terminal Tyr 110. Interestingly, oleic acid and particularly OL-GALs proved to potently inhibit MMP-13. OL-GAL was found as potent as galardin ( K i equal to 1.8 nM for OL-GAL and 1.45 nM for GAL) and selectivity for that MMP was attained (2–3 log orders of difference in inhibitory potency as compared to other MMPs). Molecular modeling studies indicated that oleic acid could be accommodated within S′ 1 pocket of MMP-13 with carboxylic acid chelating Zn ion. OL-GAL also occupied such pocket but hydroxamic acid did not interact with Zn but instead was located at 2.8 Å from Tyr 176. Since these derivatives retained, as their oleic acid original counterpart, the capacity to inhibit the amidolytic activity of HLE and plasmin as well as to decrease HLE- and plasmin-mediated pro MMP-3 activation, they might be of therapeutic value to control proteolytic cascades in chronic inflammatory disorders.

Proteolytic cleavage of annexin 1 by human leukocyte elastase

Annexin 1 has been shown to participate through its unique N-terminal domain in the recruitment and activation of leukocytes at sites of inflammation. Peptides derived from this domain are true mimetics of the annexin 1 action in all inflammation models tested and most likely serve as the active entities generated at sites of inflammation. To elucidate mechanisms underlying peptide generation we used isolated blood leukocytes and endothelial cell monolayers. We show that following endothelial adhesion, annexin 1 was externalized from leukocytes and rapidly cleaved. Addition of purified annexin 1 to degranulating leukocytes resulted in the truncation of annexin 1, which seemed to depend on the proteolytic activity of human leukocyte elastase (HLE). The capacity of elastase to proteolytically cleave annexin 1 was confirmed using both purified annexin 1 and HLE. The identification of annexin 1 as a substrate for HLE supports the model in which annexin 1 participates in regulating leukocyte emigration into inflamed tissue through N-terminal peptides generated at inflammatory sites.

Fucoidan a sulfated polysaccharide from brown algae is a potent modulator of connective tissue proteolysis

Fucoidans are sulfated fucosylated polymers from brown algae cell wall that exhibit some heparin/heparan sulfate properties. We previously demonstrated that these polysaccharides were able in vitro to stimulate dermal fibroblast proliferation and extracellular matrix deposition. Here, we investigated the action of a 16 kDa fucoidan fraction on parameters involved in connective tissue breakdown. This fucoidan is able to inhibit gelatinase A secretion and stromelysin 1 induction by interleukin-1β on dermal fibroblasts in culture. Furthermore, we observed that fucoidan increases the rate of association of MMPs with their specific inhibitors namely TIMPs. Using tissue sections of human skin in ex vivo experiments, we evidenced that this polysaccharide was able to minimize human leukocyte elastase activity resulting in the protection of human skin elastic fiber network against the enzymatic proteolysis due to this serine proteinase. These results suggested that fucoidan could be used for treating some inflammatory pathologies in which uncontrolled extracellular matrix degradation takes place.

Antiprotease Function of Airway Secretions in Purulent Tracheobronchitis

Unopposed activity of the serine protease, human leukocyte elastase (HLE), is detectable in the airways of patients with purulent tracheobronchitis. The aim of this study was to assess the compartmentalization of HLE activity in the liquid sol phase and the solid gel phase of airway secretions. Seventy samples of tracheobrochial aspirates were obtained from patients who had hypersecretion and were receiving mechanical ventilation. Samples were separated into sol and gel ("mucous pellet") phases, and HLE activity was measured using chromogenic substrate degradation. HLE was eluted from the mucous pellet using hypertonic saline solution, 1 mol/L, or bovine pancreatic deoxyribonuclease (DNase), 16 micromol/L. HLE activity partitioned between the sol and gel phases of the secretions, with most of the activity present in the gel phase (32:1 ratio of gel to sol HLE activity). The activity of HLE was 95% inhibited when bound to the gel phase, but activity appeared to be largely restored after elution from the gel phase. The gel phase was capable of binding additional exogenous HLE, and its binding capacity for exogenous HLE was not saturated by concentrations that exceeded the highest clinically relevant HLE levels (1.1 mg/mL). Hypertonic saline solution and DNase I efficiently liberated endogenous and exogenous gel phase-bound HLE activity, suggesting that electrostatic bonds and DNA, respectively, play important roles in binding HLE to the gel phase. The solid phase of airway secretions is a more important modulator of elastase-antielastase balance than has been previously recognized.

37-Kilodalton/83-Kilodalton RNase L Isoform Ratio in Peripheral Blood Mononuclear Cells: Analytical Performance and Relevance for Chronic Fatigue Syndrome

A French group has reported results (5) supporting the use of the RNase L 37-kDa/83-kDa ratio (37/83 R) in peripheral blood mononuclear cells (PBMC) as a diagnostic test for chronic fatigue syndrome (CFS). More recently (6), the same group cautioned about the diagnostic value of the 37/83 R, based on a small patient follow-up study which was likely to indicate analytical variability among duplicate assays, lack of reproducibility over time, and a weak correlation with the multidimensional fatigue inventory (MFI) score. Because of our long-term experience with this assay, we would like to offer some comments. First, we tested the analytical performance of the 37/83 R assay according to CLSI (formerly NCCLS) procedure EP5-A (4), with control samples at three different levels made of extracts of the monocytic U937 cell line spiked with various concentrations of recombinant RNase L. The guideline protocol involves assaying the samples in duplicate twice daily over a total period of 20 days. The results summarized in Table 1 indicate that both within- and between-run variation does not exceed 13%. In another series of experiments, we assayed eight patient samples in duplicate (average 37/83 R ranging from 0.5 to 245). Although in accordance with the NCCLS protocol results, the variation did not exceed 12% for samples with 37/83 R levels up to 20, and it rose significantly to 30% and more for samples with 37/83 R levels above 20. This should be expected, because beyond this level, more than 70% of the 83-kDa isoform is cleaved, and consequently, the faint 83-kDa band is difficult to scan with accuracy. Thus, in our opinion, the lower level of correlation between the duplicate assay results observed with the CFS group versus those with the controls (6) reflects the prevalence of high 37/83 R levels in the CFS group rather than a low test reproducibility as claimed by these authors. This is further supported by the good correlation found for the control group (r 0.95). During validation, the lowest detectable ratio measured with a sample containing the 83kDa isoform only was estimated (3 independent experiments with 26 replicates each) to be 0.13 0.06 (average three standard deviations). Thus, the clinical cutoff ratio of 0.4 found by the authors (5, 6) to best discriminate CFS patients from controls falls within the measurable range. Second, although long-term unexplained fatigue is a hallmark symptom, CFS is a complex clinical condition, and other important symptoms reflect an exacerbated inflammatory response. Because the inflammatory protease elastase has been shown to be responsible for the cleavage of 83-kDa RNase L into the 37-kDa isoform (2), one would expect the 37/83 R assay to reflect inflammatory activity in the immune system. In a recent study, we compared the 37/83 R with human leukocyte elastase activity (Molecular Probes kit) with 52 CFS PBMC samples. The correlation was highly significant (r 2 0.76; P 0.001), supporting this proposal. In our opinion, both fatigue and RNase L cleavage result from similar dysfunctions in the interferon-activated pathways that occur upstream of RNase L activation and exert their effects on different targets (3). Consequently, we do not feel particularly concerned by the weak correlations observed between the MFI and 37/83 R (6). The poor reproducibility of the test results over time (6) might reflect changes in the immune inflammatory status rather than in fatigue. In particular, the authors do not provide any information concerning the drugs CFS patient might have been using during the longitudinal study, which is critical as some drugs may act on the inflammatory response. Finally, by combining the results of both studies by this group (5, 6) with ours (1), which included 57 CFS patients and 28 healthy controls, we note that both diagnostic sensitivity (76%) and specificity (65%) remain attractive for such a poorly defined pathological condition.

Protective effect of a vegetable extract from Lupinus albus (LU 105) on human gingival elastic fibers degradation by human leukocyte elastase.

The aim of this study was to determine if a vegetable extract from seeds of Lupinus albus (LU 105) has the capacity to inhibit human leukocyte elastase and/or protect gingival elastic fibers against proteolytic degradation. LU105 was extracted from seeds of L albus and is freely soluble in water. In this study the ex-vivo elastolytic activity of human leukocyte elastase and the potential inhibitory effect of LU 105 were determined using human gingival cryostat tissue sections and computerized morphometric analysis. The gingival tissue sections pre-treated or not with LU 105 were submitted to the action of human leukocyte elastase or submitted to the simultaneous action of human leukocyte elastase and LU 105, and then analyzed using automated image analysis. In such conditions, LU 105 at 0.1%, 0.01%, and 0.001% developed a dose dependent protection of gingival elastic fibers against enzymatic proteolysis due to human leukocyte elastase, and LU 105 at 0.1% or 0.01% was able to inhibit the elastolytic activity of leukocyte elastase itself. It is proposed that LU 105 is an option for the treatment of gingival inflammation in which leukocyte elastase is involved.

Biochemical and pharmacological characterization of 2-(9-(2-piperidinoethoxy)-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yloxymethyl)-4-(1-methylethyl)-6-methoxy-1,2-benzisothiazol-3(2H)-one-1,1-dioxide (SSR69071), a novel, orally active elastase inhibitor.

Human leukocyte elastase (HLE) is a proteinase capable of degrading a variety of proteins. Under normal circumstances, the proteolytic activity of HLE is effectively controlled by its natural inhibitors. However, an imbalance between elastase and its endogenous inhibitors may result in several pathophysiological states such as chronic obstructive pulmonary disease, asthma, emphysema, cystic fibrosis, and chronic inflammatory diseases. It is anticipated that an orally active HLE inhibitor could be useful for the treatment of these diseases. 2-(9-(2-Piperidinoethoxy)-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yloxymethyl)-4-(1-methylethyl)-6-methoxy-1,2-benzisothiazol-3(2H)-one-1,1-dioxide (SSR69071) is a potent inhibitor of HLE, with the inhibition constant (K(i)) and the constant for inactivation process (k(on)) being 0.0168 +/- 0.0014 nM and 0.183 +/- 0.013 10(6)/mol sr, respectively. The dissociation rate constant, k(off), was 3.11 + 0.37 10(-6)/s. SSR69071 displays a higher affinity for human elastase than for rat (K(i) = 3 nM), mouse (K(i) = 1.8 nM), and rabbit (K(i) = 58 nM) elastases. Bronchoalveolar lavage fluid from mice orally treated with SSR69071 inhibits HLE (ex vivo), and in this model, SSR69071 has a dose-dependent efficacy with an ED(50) = 10.5 mg/kg p.o. SSR69071 decreases significantly the acute lung hemorrhage induced by HLE (ED(50) = 2.8 mg/kg p.o.) in mice. Furthermore, SSR69071 prevents carrageenan- (ED(30) = 2.2 mg/kg) and HLE-induced (ED(30) = 2.7 mg/kg) paw edema in rats after p.o. administration. In conclusion, SSR69071 is a selective, orally active, and potent inhibitor of HLE with good penetration in respiratory tissues.

The expression of secretory leukocyte protease inhibitor (SLPI) in the Fallopian tube: SLPI protects the acrosome reaction of sperm from inhibitory effects of elastase

Secretory leukocyte protease inhibitor (SLPI) is a protein found in various fluids, including parotid secretions, cervical mucus, seminal plasma and ascites, and is a potent inhibitor of human leukocyte elastase activity. The objective of the study was 2-fold, to evaluate (i) the presence of SLPI in the Fallopian tube, and (ii) the effect of SLPI on the acrosome reaction of sperm. Western blot analysis revealed that SLPI protein was detected as a 12 kDa band in the isthmus, ampulla and infundibulum of the Fallopian tube. Immunohistochemistry using an anti-SLPI polyclonal antibody revealed positive staining of epithelial cells in the Fallopian tube. RT-PCR demonstrated that SLPI transcripts were expressed in the Fallopian tube. To determine the function of SLPI in the Fallopian tube, the effects of SLPI and elastase on the sperm acrosome reaction were examined. SLPI prevented the reduction of the acrosome reaction by elastase in a dose-dependent manner. The present findings suggest that SLPI in the Fallopian tube contributes to sperm-oocyte interaction.

Inhibitory Effects of 150 Plant Extracts on Elastase Activity, and Their Anti‐inflammatory Effects

The inhibitory effects of 150 medicinal plants on elastase activity were investigated. Among the 150 plants, six plant extracts (final concentration 1 mg/ml in methanol) exhibited more than 65% of inhibition of elastase activity. The inhibitory effects of six active plants, including Areca catechu (IC50, 42.4 mug/ml), Cinnamonum cassia (IC50, 208.7 mug/ml), Myristica fragrans (IC50, 284.1 mug/ml), Curcuma longa (IC50, 398.4 mug/ml), Alpinia katsumadai (IC50, 465.7 mug/ml) and Dryopteris cassirrhizoma (IC50, 714.4 mug/ml) on the activity of human leukocyte elastase, hyaluronidase and lipid peroxidation were examined. In the lipid peroxidation assay, using the TBA method, three of the six plants, including Curcuma longa (IC50, 45.5 mug/ml), Areca catechu (IC50, 51.0 mug/ml) and Alpinia katsumadai (IC50, 116.3 mug/ml) exhibited more than 70% inhibition at the concentration of 1 mug/ml, but only one plant, Areca catechu (IC50, 563 mug/ml) showed high inhibitory effect on hyaluronidase activity. The results suggest that medicinal plants showing several biological activities may be potent inhibitors of the anti-ageing process in skin. This property might be useful for application in cosmetics.

Inhibition of human leukocyte elastase activity by chondroitin sulfates

Chondroitin sulfates with different properties and devoid of appreciable anticoagulant activity were evaluated for their capacity to inhibit human leukocyte elastase activity in vitro by using a chromogenic substrate. Fractions with various mass and the same charge density were tested. The chondroitin sulfates with a molecular mass greater than about 2000 inhibited human leukocyte elastase activity to the same extent, whilst the fractions with a molecular mass of 1960 and 1020 were much less effective. The percentage inhibition of human leukocyte elastase activity increased based on the charge density of chondroitin sulfates. In particular, the inhibition of the enzymatic activity decreased with the percentage of non-sulfated disaccharide and increased with the amount of disaccharide-2,6-disulfated.

Inhibition of human leukocyte elastase activity by heparins: influence of charge density

Heparins with different structures and physico-chemical properties were evaluated for their capacity to inhibit human leukocyte elastase activity in vitro by using a chromogenic substrate. Heparin from bovine intestinal mucosa and heparan sulfate from bovine spleen were extracted and purified, and their purity, structures, and physico-chemical properties were evaluated. Slow moving and fast moving heparin species were obtained by selective precipitation as barium salt, and partially desulfated and re- N-sulfated heparin was produced by chemical modifications. Heparins with different molecular mass (from 950 to 7820), narrow polydispersity and the same charge density were produced by a chemical depolymerization process in the presence of free radicals, and further gel-permeation chromatography. Heparins strongly inhibit elastase activity, and there is a significant linear dependence between charge density (sulfate-to-carboxyl ratio) and enzymatic activity. We also found a significant linear correlation between the percentage of N-sulfate groups and increased inhibition of elastase activity and between the percentage of iduronic acid and enzymatic activity. Heparin samples with a M r greater than about 2000–3000 inhibit the HLE activity to the same extent (about 59%) whilst two fractions with a M r of 1530 (29% inhibition of HLE activity) and 950 (4% inhibition of HLE activity) have less capacity to produce a decrease in the enzymatic activity.

The inhibitory effects of antirheumatic drugs on the activity of human leukocyte elastase and cathepsin G.

The serine proteinases elastase and cathepsin G from polymorphonuclear granulocytes play a critical role in articular cartilage degradation, not only as proteolytic enzymes able to degrade the extracellular matrix but also by additionally modulating the level of active matrix metalloproteinases, key enzymes of the proteolytic destruction of cartilage during rheumatoid arthritis. The aim of our study was to examine whether anti-inflammatory drugs and selected compounds inhibited elastase and cathepsin G, and also to determine whether it is necessary to use a highly purified elastase preparation to screen drugs for their ability to block the activity of this enzyme. Eglin C and the glycosaminoglycan-peptide complex DAK-16, at concentrations ranging from 10(-9) to 10(-4) M, dose-dependently inhibited elastase and cathepsin G while the nonsteroidal anti-inflammatory drugs oxyphenbutazone, phenylbutazone, sulfinpyrazone and diclofenac-Na required high concentrations to demonstrate some inhibitory effects on the activity of both enzymes. None of the other anti-inflammatory drugs tested at a concentration of 10(-4) M such as acetylsalicylic acid, dexamethasone, indomethacin, ketoprofen, naproxen, oxaceprol, pirprofen and tiaprofenic acid demonstrated any marked inhibitory activity on either of these proteinases. Only a few drugs, when dosed therapeutically, achieved synovial fluid concentrations sufficient to inhibit the activities of both proteinases. The antirheumatic drugs demonstrated similar inhibition profiles in purified or partially purified elastase preparations. Thus the leukocyte extract containing the partially purified elastase and cathepsin G which can be rapidly and easily prepared at low costs appears to be an efficient mean of screening potentially new therapeutic agents for their ability to inhibit leukocyte elastase and cathepsin G.

Proteoglycan-degrading activity of human stromelysin-1 and leukocyte elastase in rabbit joints. Quantitation of proteoglycan and a stromelysin-induced HABR fragment of aggrecan in synovial fluid and cartilage.

The objective of this study was to compare the specificity and potency of recombinant human SLN-1 (rhSLN) and human leukocyte elastase (HLE) as proteoglycan (PG)-degrading enzymes after intraarticular injection into rabbits. Another objective was to evaluate the elicitation of a rhSLN-induced hyaluronan-binding region (HABR) fragment from rabbit aggrecan in joints using a polyclonal antiserum (anti-FVDIPEN) against the synthetic peptide, Phe-Val-Asp-Ile-Pro-Glu-Asn (FVDIPEN). The intraarticular injection of either activated rhSLN or HLE resulted in enzyme-specific quantitative release of PG fragments into synovial fluid. Based on the criteria used herein, HLE appears to be a more potent PG-degrading enzyme than SLN. Intraarticular injection of rhSLN also resulted in time- and dose-dependent release of a new HABR fragment of aggrecan (HABR-FMDIPEN) into both articular cartilage and synovial fluid. HABR-FVDIPEN is likely to be a good marker of matrix metalloproteinase (MMP)-induced degradation of aggrecan.

Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases.

Serine proteinases of human polymorphonuclear neutrophils play an important role in neutrophil-mediated proteolytic events; however, the non-oxidative mechanisms by which the cells can degrade extracellular matrix in the presence of proteinase inhibitors have not been elucidated. Herein, we provide the first report that human neutrophils express persistently active cell surface-bound human leukocyte elastase and cathepsin G on their cell surface. Unstimulated neutrophils have minimal cell surface expression of these enzymes; however, phorbol ester induces a 30-fold increase. While exposure of neutrophils to chemoattractants (fMLP and C5a) stimulates modest (two- to threefold) increases in cell surface expression of serine proteinases, priming with concentrations of lipopolysaccharide as low as 100 fg/ml leads to striking (up to 10-fold) increase in chemoattractant-induced cell surface expression, even in the presence of serum proteins. LPS-primed and fMLP-stimulated neutrophils have approximately 100 ng of cell surface human leukocyte elastase activity per 10(6) cells. Cell surface-bound human leukocyte elastase is catalytically active, yet is remarkably resistant to inhibition by naturally occurring proteinase inhibitors. These data indicate that binding of serine proteinases to the cell surface focuses and preserves their catalytic activity, even in the presence of proteinase inhibitors. Upregulated expression of persistently active cell surface-bound serine proteinases on activated neutrophils provides a novel mechanism to facilitate their egress from the vasculature, penetration of tissue barriers, and recruitment into sites of inflammation. Dysregulation of the cell surface expression of these enzymes has the potential to cause tissue destruction during inflammation.

Inhibition of neutrophil elastase by mucus glycoprotein.

A number of structurally diverse polyanions have been found to inhibit human leukocyte elastase (HLE) activity. The purpose of this study was to examine the effects of mucus glycoprotein (mucin), one of the most plentiful high molecular weight polyanions in the respiratory tracts, on HLE activity. Human airway mucin and bovine submaxillary mucin at concentrations of 0.4 to 2.8 mg/ml both markedly inhibited the elastolytic activity of 50 nM HLE, with maximum inhibition approaching 90%. The degree of inhibition was the same regardless of whether the mucin, elastase, and elastin were simultaneously combined or whether the mucin was added to elastase 20 min prior to adding elastin, indicating that mucin is a rapid-acting inhibitor. The shape of the inhibition curve resembled that of curves obtained using heparin and HLE. Mucin had little inhibitory effect on pancreatic elastase, which is structurally related to but less cationic than HLE. The inhibition of HLE by mucin was blocked by 1 M NaCl. Removal of sulfate esters by acid-catalyzed solvolysis markedly reduced the inhibitory effect of bovine submaxillary mucin. These results indicate that inhibition of HLE by mucin involves binding of the positively charged HLE molecules to the negatively charged sulfated carbohydrates in the mucin. Mucin was also found to substantially reduce the antiprotease activity of secretory leukocyte protease inhibitor, a low molecular weight cationic protein known to bind to mucin.

Activation of pro-urokinase by cathepsin G in the presence of glucosaminoglycans

Summary The effect of extracellular matrix glycosaminoglycans (GAGs) on the activity of cathepsin G (Cat G) was investigated. Heparin inhibited the amidolytic activity of Cat G with some peptidyl substrates, whereas it enhanced the activity with others. The effect on Cat G activity was observed with high M r heparin as well as with fractions of relatively low M r . This is in contrast to the effect of heparin on the activity of human leukocyte elastase (HLE) where the effect was observed only with high M r fractions. The Cat G-mediated activation of pro-u-PA was also investigated. In agreement with previous observations (Learmonth et al. Fibrinolysis 1992; 6 (suppl 4): 113–116) we found that Cat G cleaved pro-u-PA primarily in the epidermal growth factor-like domain, and secondarily at, or close to the activation site. Our results showed that the interaction between Cat G and pro-u-PA was strongly enhanced by heparin, and that 75% conversion into an active u-PA form could be obtained. Stimulation of Cat G-catalyzed activation of pro-u-PA was also observed with heparan sulphate and chondroitin sulphate A, B and C. The effect of various fractions of heparin was investigated and shown to depend strongly on the M r with little or no stimulation of the low-M r fractions. We propose that a substantial amount of active u-PA with impaired receptor binding may accumulate under some conditions as a result of influx and activation of neutrophils.

Effects of urinary trypsin inhibitor on the invasion of reconstituted basement membranes by ovarian cancer cells

Using the human ovarian cancer cell line HOC-1, we investigated the effects of urinary trypsin inhibitor (UTI) purified from human urine and its related synthetic peptides on the invasive potential of cancer cells in an in vitro assay. Invasiveness of tumor cells was determined using a modified Boyden chamber and a reconstituted basement membrane Matrigel. Three peptides (peptide 1, peptide 2, and peptide 3), representing sequences within UTI, were synthesized. HOC-1 cells showed detectable and reproducible levels of expression of surface urokinase-type plasminogen activator (uPA) and plasminogen/plasmin by cell ELISAs and enzyme assays. UTI was found to strongly inhibit plasmin and human leukocyte elastase (HLE). Peptide 2 and peptide 3 specifically inhibit HLE and plasmin activity respectively. Peptide 1 has essentially no inhibitory activity. Treatment with UTI and peptide 3 reduces the incidence of invasion, whereas peptide 1 and peptide 2 do not affect invasion. The inhibitory effect on cell invasion is dose-dependent. The proteolytic enzyme plasmin may be involved in human ovarian cancer invasion in extracellular matrix degradation, and the use of UTI and peptide 3 that inhibits plasmin specifically reduces invasion by tumor cells.

Effect of type-1 plasminogen activator inhibitor on human leukocyte elastase

When human leukocyte elastase (HLE) activity (1.0 μg/ml) was analysed in the presence of PAI-I (0–15.0 μg/ml), HLE activity, measured with the low molecular weight paranitroanilide substrate L-pyroglutamyl-prolyl-L-valine-p-nitroanilide was increased time and dose dependently (a plateau of stimulation was reached after 30 minutes) with a simultaneous decrease in PAI-1 inhibitory activity. This effect was neither influenced by the presence of vitronectin not heparin. When PAI-1 was converted into its latent form by incubation for 48 hours at 37°C or incubated with an excess of recombinant t-PA to convert free PAI-1 into t-PA-PAI-1 complexes, the stimulatory effect of both the latent and the complexed form of PAI-1 was significantly greater than that of the active form. Analysing HLE PAI-1 interaction on a molecular level using SDS-PAGE, no SDS stable complex formation between HLE and PAI-1 could be observed but lower molecular weight cleavage products of PAI-1 were generated. The stimulatory effect of PAI-1 on HLE activity was not restricted to the low molecular weight pNA-substrate but was also revealed using a natural substrate assay (bovine neck ligament elastin solubilization). Therefore interaction of HLE and PAI-1 seems not to be restricted just to decrease PAI-1 activity but would simultaneously also increase HLE activity, thereby supporting enzymatic activity necessary for migration of leukocytes, dissolution of blood clots and tissue remodelling.