Optimisation of a human neutrophil elastase assay and investigation of the effect of sesquiterpene lactones.

Neutrophil granulocytes are effector cells in innate and humoral immunity. They are involved in inflammatory processes by releasing pro-inflammatory enzymes, such as the human neutrophil elastase (HNE). We here report an optimisation of an HNE release assay using microplates. Special attention has been directed to overcome the often observed activation of neutrophils during isolation from fresh blood. This so-called basal stimulation can take place without addition of external stimulants and can cause severe problems during the assay. We demonstrated that bovine serum albumin (BSA), lipopolysaccharide (LPS), use of different blood donors, heparin and ethylenediaminetetraacetic acid (EDTA) do not cause basal stimulation, but may be due to mechanical stress and the immune system of the blood donor. Here, the number of eosinophils may play a role in the induction of activation. Basal stimulation was overcome when a hypertonic solution, such as sucrose- with N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid (HEPES) buffer, was used during centrifugation and the isolated granulocytes were left in phosphate buffered saline (PBS) for 30 min before stimulation. Sesquiterpene lactones (SLs), known for their anti-inflammatory activity were used for evaluation of the assay and were observed to inhibit HNE release at micromolar concentrations. Whereas N-formyl-methionyl-leucylphenylalanine (fMLP), platelet-activating factor (PAF) and basal stimulation resulted in similar IC50 values, phorbol-12-myristate-13-acetate (PMA) as a stimulant needed higher concentrations of SLs. The molecular inhibitory mechanism of SLs is discussed.     

Discriminating between the activities of human neutrophil elastase and proteinase 3 using serpin-derived fluorogenic substrates

Human neutrophil elastase (HNE) has long been linked to the pathology of a variety of inflammatory diseases and therefore is a potential target for therapeutic intervention. At least two other serine proteases, proteinase 3 (Pr3) and cathepsin G, are stored within the same neutrophil primary granules as HNE and are released from the cell at the same time at inflammatory sites. HNE and Pr3 are structurally and functionally very similar, and no substrate is currently available that is preferentially cleaved by Pr3 rather than HNE. Discrimination between these two proteases is the first step in elucidating their relative contributions to the development and spread of inflammatory diseases. Therefore, we have prepared new fluorescent peptidyl substrates derived from natural target proteins of the serpin family. This was done because serpins are rapidly cleaved within their reactive site loop whether they act as protease substrates or inhibitors. The hydrolysis of peptide substrates reflects the specificity of the parent serpin including those from alpha-1-protease inhibitor and monocyte neutrophil elastase inhibitor, two potent inhibitors of elastase and Pr3. More specific substrates for these proteases were derived from the reactive site loop of plasminogen activator inhibitor 1, proteinase inhibitors 6 and 9, and from the related viral cytokine response modifier A (CrmA). This improved specificity was obtained by using a cysteinyl residue at P1 for Pr3 and an Ile residue for HNE and because of occupation of protease S' subsites. These substrates enabled us to quantify nanomolar concentrations of HNE and Pr3 that were free in solution or bound at the neutrophil surface. As membrane-bound proteases resist inhibition by endogenous inhibitors, measuring their activity at the surface of neutrophils may be a great help in understanding their role during inflammation.     

Direct interaction of ONO-5046 with human neutrophil elastase through ¹H NMR and molecular docking

Human neutrophil elastase (HNE) has been implicated as a major contributor in the pathogenesis of diseases, such as pulmonary emphysema, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and other inflammatory diseases. Therefore, searching for appropriate and potential human neutrophil elastase inhibitors (HNEI) that would restore the balance between the free enzyme and the endogenous inhibitors would be of therapeutic interest. ONO-5046 is the first specific HNEI to improve respiratory function and protect lung tissues against various lung injuries. However, the mechanism of ONO-5046 to HNE is still unclear. In this study, the binding properties of ONO-5046 were investigated through (1)H NMR, molecular docking, and bioassay methods to understand the effect of ONO-5046 to HNE. The proton spin-lattice relaxation rate and molecular rotational correlation time results indicated that ONO-5046 has higher affinity with HNE. The molecular docking study showed that ONO-5046 is perfectly matched for the primary enzyme specificity pocket (S1 pocket), and is tightly bound to this pocket of HNE through hydrophobic and hydrogen bonding interactions. The results of both methods were validated through analysis of the HNE inhibitory activity bioassay of ONO-5046 with an IC(50) value of 87.05 nM. Our data suggested that ONO-5046 could bind to HNE through direct interaction, and that molecular docking and NMR methods are valid approaches to survey new HNEI.     

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.     

Human neutrophil-derived elastase induces airway smooth muscle cell proliferation

Neutrophils and their derived elastase are abundant in chronic inflammatory responses of asthma. This study aimed to investigate the mitogenic effect of elastase on airway smooth muscle (ASM) cells and the implicated signal transduction pathway. Near confluent cultured human ASM cells were treated with human neutrophil elastase (HNE, 0.01 to 0.5 microg/ml) or vehicle for 24 hours with or without extracellular signal-regulated kinase (ERK) inhibitor (PD98059, 30 microM), p38 kinase inhibitor (SB203580, 10 microM) or elastase inhibitor II (100 microg/ml). The ASM cell numbers were counted by a hemocytometer and DNA synthesis was assessed by flowcytometry. Western blots analysis for the expression of ERK, p38 and cyclin D1 was determined. HNE dose-dependently increased ASM cell numbers and the percentage of cells entering S-phase of cell cycle. This response was abolished by neutrophil elastase inhibitors and attenuated by PD98059, but not SB203580. HNE increased ERK phosphorylation and cyclin D1 expression. Pretreatment with PD98059 significantly inhibited elastase-induced cyclin D1 activity. The increased ASM cellular gap and cell shape change by proteolytic activity of HNE may be contributory to ERK activation and therefore cell proliferation. Our results demonstrate that HNE is mitogenic for ASM cells by increasing cyclin D1 activity through ERK signaling pathway.     

Design, synthesis and evaluation of N-benzoylindazole derivatives and analogues as inhibitors of human neutrophil elastase

Human neutrophil elastase (HNE) plays an important role in tumour invasion and inflammation. A series of N-benzoylindazoles was synthesized and evaluated for their ability to inhibit HNE. We found that this scaffold is appropriate for HNE inhibitors and that the benzoyl fragment at position 1 is essential for activity. The most active compounds inhibited HNE activity with IC?? values in the submicromolar range. Furthermore, docking studies indicated that the geometry of an inhibitor within the binding site and energetics of Michaelis complex formation were key factors influencing the inhibitor's biological activity. Thus, N-benzoylindazole derivatives and their analogs represent novel structural templates that can be utilized for further development of efficacious HNE inhibitors.     

Human neutrophil elastase: mediator and therapeutic target in atherosclerosis

Human neutrophil elastase (HNE) is present within atherosclerotic plaques where it contributes to matrix degradation and weakening of the vessel wall associated with the complications of aneurysm formation and plaque rupture. It is joined by other extracellular proteases in these actions but the broad range of substrates and potency of HNE coupled with the potential for rapid increases in HNE activity associated with neutrophil degranulation in acute coronary syndromes single this disruptive protease out as therapeutic target in atherosclerotic disease. This review summarises the role of HNE in neutrophil-mediated endothelial injury and the evidence for HNE as a mediator of atherosclerotic plaque development. The therapeutic potential of HNE neutralising antiproteases, alpha-1-antitrypsin and elafin, in atherosclerosis, is discussed.     

Determination of proteinase 3-alpha 1-antitrypsin complexes in inflammatory fluids.

Physiological inhibitors were tested for their in vitro interaction with neutrophil proteinase 3 (PR3). The major plasma proteinase inhibitor of PR3 is alpha 1AT. We have developed a radioimmunoassay (RIA) for quantitative detection of PR3-alpha 1AT complexes formed in vivo in inflammatory exudates such as synovial fluid and plasma from patients with sepsis. Levels of PR3-alpha 1AT complexes correlated significantly with levels of human neutrophil elastase (HNE)-alpha 1AT complexes. Thus, in vivo alpha 1AT not only protects against excessive HNE activity, but also against excessive PR3 activity.     

Biochemical and Immunological Identification of Human Neutrophil Elastase on Nitrocellulose Membranes

Human neutrophil elastase (HNE) was analyzed for protein(s), antibody staining and activity staining, on lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis followed by Western blotting. The HNE activity, which was identified with N-acetyl-D,L-alanine α-naphthyl ester as substrate, was well preserved in the presence of 0.1% LDS at 4 C during electrophoresis. As little as 0.1 μg HNE was required for the activity staining. The HNE appeared to be three peptides having a major band at mass ratio 27,000, a second major band at mass ratio 28,000 with a minor protein band at mass ratio 29,000. On transfer to nitrocellulose, the mass ratio 28,000 band displayed poor immunoreactivity. This was the second most dense band with highest enzymatic staining. This procedure is a useful method and analytical tool to determine the correlation of enzymatically active proteins, subunits and immunoreactive protein(s) of elastase from various sources, including neutrophils.     

Degradation of plasmodial antigens by human neutrophil elastase

Human neutrophil elastase (HNE) has been well-studied with respect to its role in pathologic states, but less is known about the physiologic functions of this important granulocyte enzyme. In the present study, we show that HNE can degrade the major circumsporozoite protein of the infective (sporozoite) stage of Plasmodium vivax malaria, and that this enzyme can also interfere with the cytoadherence of human E infected with Plasmodium falciparum (strain K+ FMG-FCR3) (IE). Cytoadherence reactions are not only blocked by treatment of IE with as little as 10 fg HNE/IE, but already adherent IE are also removed by the enzyme. Normal E surface Ag are not extensively destroyed by these doses of HNE. This suggests that the effect of HNE on cytoadherence is selective and probably due to degradation of the malarial Ag exported to the IE surface and responsible for the formation of "recognition knobs" upon which the cytoadherence reaction depends. This conclusion, in turn, was supported by the results of Western blot analysis showing that HNE degrades a high m.w. Ag found exclusively in membrane extracts of IE. Our results suggest that one physiologic role of HNE may be degradation of parasitic antigens during host defense against malaria.     

Mechanism for the increased permeability in endothelial monolayers induced by elastase

The aim of this study was to investigate the mechanism for the increase in endothelial permeability induced by human neutrophil elastase (HNE). Pretreatment of bovine pulmonary artery endothelial cells (BPAEC) with HNE(0-30 mug/ml) for 1 h produced a concentration dependent increase in (125)I-albumin clearance. The effect was reversible and was not due to cytolysis. Pretreatment of BPAEC with sodium tungstate, which depletes xanthine oxidase, or with oxypurinol, did not prevent HNE induced increased permeability. Heparin, which neutralizes the cationic charge of HNE, also had no protective effect. Pretreatment with heat inactivated HNE, which still had positive charge sites, did not result in increased endothelial permeability. Also, ONO-5046, a novel specific inhibitor of HNE, did prevent increased permeability. These results suggest that elastase increases endothelial permeability mainly through its proteolytic effects.     

Biochemical and immunological identification of human neutrophil elastase on nitrocellulose membranes.

Human neutrophil elastase (HNE) was analyzed for protein(s), antibody staining and activity staining, on lithium dodecyl sulfate (LDS) polyacrylamide gel electrophoresis followed by Western blotting. The HNE activity, which was identified with N-acetyl-D,L-alanine alpha-naphthyl ester as substrate, was well preserved in the presence of 0.1% LDS at 4 C during electrophoresis. As little as 0.1 microgram HNE was required for the activity staining. The HNE appeared to be three peptides having a major band at mass ratio 27,000, a second major band at mass ratio 28,000 with a minor protein band at mass ratio 29,000. On transfer to nitrocellulose, the mass ratio 28,000 band displayed poor immunoreactivity. This was the second most dense band with highest enzymatic staining. This procedure is a useful method and analytical tool to determine the correlation of enzymatically active proteins, subunits and immunoreactive protein(s) of elastase from various sources, including neutrophils.     

Non-peptidic inhibitors of human neutrophil elastase: The design and synthesis of sulfonanilide-containing inhibitors

A novel series of pivaloyloxy benzene derivatives has been identified as potent and selective human neutrophil elastase (HNE) inhibitors. Convergent syntheses were developed in order to identify the inhibitors which are intravenously effective in an animal model. A compound of particular interest is the sulfonanilide-containing analogues. Structure-activity relationships are discussed. Structural requirements for metabolic stabilization are also discussed.     

Cationic neutrophil proteins increase transendothelial albumin movement

Neutrophils play a role in the development of pulmonary edema in many models of the adult respiratory distress syndrome, but the mechanism of their action is not completely understood. We asked whether two neutrophil secretory products, human neutrophil cationic protein (NCP) and human neutrophil elastase (HNE), would nonenzymatically alter the movement of albumin across a cultured endothelial monolayer. Both enzymes were inactivated by heating before use. HNE was additionally enzymatically inactivated with a chloromethylketone oligopeptide (CMK) inhibitor and with alpha 1-proteinase inhibitor (alpha 1-PI). Heated NCP, heated HNE, and CMK-complexed HNE all increased transendothelial albumin transfer. The cation protamine also increased albumin transfer across the endothelium and this increase was blocked by heparin. Alpha 1-PI and fetal bovine serum also prevented the cationic proteins from increasing albumin transfer. Using the release of lactate dehydrogenase as a marker of cytotoxicity, heated HNE was toxic to endothelial cells, heated NCP had only minimal toxicity, and protamine had no toxicity. Changes in endothelial cell shape with gap formation was seen after exposure to both heated HNE and heated NCP. Both the cytotoxicity associated with heated HNE and the cell shape changes associated with heated NCP and heated HNE could be blocked by heparin. These results suggest that in addition to neutrophil proteases and reactive O2 molecules, neutrophil-derived cationic proteins can directly and nonenzymatically contribute to edema formation during acute inflammation.     

The association of an elastase with amyloid fibrils

The fibrils of all systemic forms of amyloid (primary, AL; secondary, AA; and hereditary, AF) that had been isolated by the water extraction procedure demonstrated elastolytic enzyme activity when examined in a specific assay using tritiated elastin. The source of this fibril-bound enzyme activity was consistent with human neutrophil elastase (HNE), since it was readily extracted by high salt solutions and inhibited by an elastase-specific chloromethyl ketone inhibitor, human alpha-1-protease inhibitor or by an antibody specific for HNE. The presence of an elastase on the amyloid fibril may suggest physiologic mechanisms of amyloid precursor protein degradation.     

Design and synthesis of peptide-based carboxylic acid-containing transition-state inhibitors of human neutrophil elastase

In our search for a new agent, human neutrophil elastase (HNE) inhibitor, for the treatment of acute respiratory failure, we rationally designed and synthesized a series of peptide-based carboxylic acid-containing transition-state inhibitors. The presence of valyl moiety is found to be essential for potent in vitro inhibitory activity and also prevention of an undesirable toxicity. Of these, compound 9m has the most potent in vivo effect on HNE-induced lung hemorrhage in hamsters.     

Bornyl (3,4,5-trihydroxy)-cinnamate--an optimized human neutrophil elastase inhibitor designed by free energy calculations

Human neutrophil elastase (HNE), a serine protease, is involved in the regulation of inflammatory processes and controlled by endogenous proteinase inhibitors. Abnormally high levels of HNE can cause degradation of healthy tissues contributing to inflammatory diseases such as rheumatoid arthritis, and also psoriasis and delayed wound healing. In continuation of our research on HNE inhibitors we have used the recently developed binding mode model for a group of cinnamic acid derivative elastase inhibitors and created bornyl (3,4,5-trihydroxy)-cinnamate. This ligand exhibited improved binding affinity predicted by means of free energy calculations. An organic synthesis scheme for the ligand was developed and its inhibitory activity was tested toward the isolated enzyme. Its IC(50) value was found to be three times lower than that of similar compounds, which is in line with the computational result showing the high potential of free energy calculations as a tool in drug development.     

Plasma Neutrophil Elastase and Elafin Imbalance Is Associated with Acute Respiratory Distress Syndrome (ARDS) Development

Background

We conducted an exploratory study of genome-wide gene expression in whole blood and found that the expression of neutrophil elastase inhibitor (PI3, elafin) was down-regulated during the early phase of ARDS. Further analyses of plasma PI3 levels revealed a rapid decrease during early ARDS development. PI3 and secretory leukocyte proteinase inhibitor (SLPI) are important low-molecular-weight proteinase inhibitors produced locally at neutrophil infiltration site in the lung. In this study, we tested the hypothesis that an imbalance between neutrophil elastase (HNE) and its inhibitors in blood is related to the development of ARDS.

Methodology/Principal Findings

PI3, SLPI, and HNE were measured in plasma samples collected from 148 ARDS patients and 63 critical ill patients at risk for ARDS (controls). Compared with the controls, the ARDS patients had higher HNE, but lower PI3, at the onset of ARDS, resulting in increased HNE/PI3 ratio (mean = 14.5; 95% CI, 10.9–19.4, P<0.0001), whereas plasma SLPI was not associated with the risk of ARDS development. Although the controls had elevated plasma PI3 and HNE, their HNE/PI3 ratio (mean = 6.5; 95% CI, 4.9–8.8) was not significantly different from the healthy individuals (mean = 3.9; 95% CI, 2.7–5.9). Before the onset (7-days period prior to ARDS diagnosis), we only observed significantly elevated HNE, but the HNE-PI3 balance remained normal. With the progress from prior to the onset of ARDS, the plasma level of PI3 declined, whereas HNE was maintained at a higher level, tilting the balance toward more HNE in the circulation as characterized by an increased HNE/PI3 ratio. In contrast, three days after ICU admission, there was a significant drop of HNE/PI3 ratio in the at-risk controls.

Conclusions/Significance

Plasma profiles of PI3, HNE, and HNE/PI3 may be useful clinical biomarkers in monitoring the development of ARDS.     

N-Acyl and N-sulfonyloxazolidine-2,4-diones are pseudo-irreversible inhibitors of serine proteases

The synthesis, inhibitory activity and mode of action of oxazolidine-2,4-diones against porcine pancreatic elastase, here used as a model for human neutrophil elastase, are reported. The nature of N-substitution at the oxazolidine-2,4-dione scaffold has large effect on the inhibitory potency against elastase. N-Acyl and N-sulfonyloxazolidine-2,4-diones emerged as potent pseudo-irreversible inhibitors, displaying high second-order rate constants for PPE inactivation. The title compounds were also shown to be potent inhibitors of human neutrophil elastase (HNE) and proteinase-3, and weak inhibitors of human cathepsin G. The results herein presented show that the oxazolidine-2,4-diones represent a new promising class of serine protease inhibitors.     

Simple phosphonic inhibitors of human neutrophil elastase

Herein, we describe the synthesis and resulting activity of a complex series of α-aminophosphonate diaryl esters as irreversible human neutrophil elastase inhibitors and their selectivity preference for human neutrophil elastase over several other serine proteases such as porcine pancreatic elastase, trypsin, and chymotrypsin. We synthesized and examined the inhibitory potency of several new simple Cbz-protected α-aminoalkylphosphonate diaryl esters that yielded several new HNE inhibitors, where one of the obtained compounds Cbz-Val^P(OC₆H₄-4-COOMe)₂ displayed an apparent second-order inhibition value at 33,015 M⁻¹ s⁻¹.