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The serine proteases released by activated polymorphonuclear neutrophils [NSPs (neutrophil serine proteases)] contribute to a variety of inflammatory lung diseases, including CF (cystic fibrosis). They are therefore key targets for the development of efficient inhibitors. Although rodent models have contributed to our understanding of several diseases, we have previously shown that they are not appropriate for testing anti-NSP therapeutic strategies [Kalupov, Brillard-Bourdet, Dade, Serrano, Wartelle, Guyot, Juliano, Moreau, Belaaouaj and Gauthier (2009) J. Biol. Chem. 284, 34084-34091). Thus NSPs must be characterized in an animal model that is much more likely to predict how therapies will act in humans in order to develop protease inhibitors as drugs. The recently developed CFTR-/- (CFTR is CF transmembrane conductance regulator) pig model is a promising alternative to the mouse model of CF [Rogers, Stoltz, Meyerholz, Ostedgaard, Rokhlina, Taft, Rogan, Pezzulo, Karp, Itani et al. (2008) Science 321, 1837-1841]. We have isolated blood neutrophils from healthy pigs and determined their responses to the bacterial pathogens Pseudomonas aeruginosa and Staphylococcus aureus, and the biochemical properties of their NSPs. We used confocal microscopy and antibodies directed against their human homologues to show that the three NSPs (elastase, protease 3 and cathepsin G) are enzymatically active and present on the surface of triggered neutrophils and NETs (neutrophil extracellular traps). All of the porcine NSPs are effectively inhibited by human NSP inhibitors. We conclude that there is a close functional resemblance between porcine and human NSPs. The pig is therefore a suitable animal model for testing new NSP inhibitors as anti-inflammatory agents in neutrophil-associated diseases such as CF.

Neutrophil serine proteases (NSPs), including elastase, proteinase 3 and cathepsin G, play critical roles in the pathogenesis of chronic inflammatory lung diseases. The release of excess NSPs leads to the destruction of lung tissue and an overexuberant, sustained inflammatory response. Antiproteases could be valuable tools for controlling these NSP-mediated inflammatory events. We have examined the capacity of trappin-2 A62L, a potent engineered inhibitor of all three NSPs, to protect human lung A549 epithelial cells from the deleterious effects of NSPs. Trappin-2 A62L, significantly inhibited the detachment of A549 cells and the degradation of the tight-junction proteins, E-cadherin, β-catenin and ZO-1, induced by each individual NSP and by activated neutrophils. Trappin-2 A62L also decreased the release of the pro-inflammatory cytokines IL-6 and IL-8 from A549 cells that had been stimulated with elastase or LPS. Trappin-2 A62D/M63L, a trappin-2 variant that has no antiprotease activity, has similar properties, suggesting that the anti-inflammatory action of trappin-2 is independent of its antiprotease activity. Interestingly, we present evidence that trappin-2 A62L, as well as wild-type trappin-2, enter A549 cells and move rapidly to the cytoplasm and nucleus, where they are likely to exert their anti-inflammatory effects. We have also demonstrated that trappin-2 A62L inhibits the early apoptosis of A549 cells mediated by NSPs. Thus, our data indicate that trappin-2 A62L is a powerful anti-protease and anti-inflammatory agent that could be used to develop a treatment for patients with inflammatory lung diseases., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases, large quantities of which are stored in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to degrade engulfed microorganisms inside phagolysosomes. Active forms of these proteases are also externalized during neutrophil activation at inflammatory sites, thus helping to regulate inflammatory and immune responses. A fraction of secreted neutrophil serine proteases (NSPs) remains bound to the external plasma membrane, where they remain enzymatically active. This protocol describes the spectrofluorometric measurement of NSP activities using sensitive ortho-aminobenzoyl-peptidyl-N-(2,4-dinitrophenyl) ethylenediamine fluorescence resonance energy transfer (FRET) substrates that fully discriminate between the three human NSPs. These are used to measure subnanomolar concentrations of free or membrane-bound NSPs in low-binding microplates and to quantify the activities of individual proteases in biological fluids. We describe the synthesis of FRET substrate, neutrophil purification, and kinetic experiments on activated neutrophils. The protocol for measuring NSP activity on the surface of activated neutrophils can be adapted to measure NSP activities in whole biological fluids. Such data clarify the contributions of individual NSPs to the development of inflammatory diseases. Ultimately, these proteases may be shown to be targets for therapeutic inhibitors.

The physiological and pathological functions of proteinase 3 (PR3) are not well understood due to its close similarity to human neutrophil elastase (HNE) and the lack of a specific inhibitor. Based on structural analysis of the active sites of PR3 and HNE, we generated mutants derived from the polyvalent inhibitor SerpinB1 (monocyte/neutrophil elastase inhibitor) that specifically inhibit PR3 and that differ from wt-SerpinB1 by only 3 or 4 residues in the reactive center loop. The rate constant of association between the best SerpinB1 mutant and PR3 is 1.4 × 10⁷ M⁻¹ · s⁻¹, which is ∼100-fold higher than that observed with wt-SerpinB1 and compares with that of α1-protease inhibitor (α1-PI) toward HNE. SerpinB1(S/DAR) is cleaved by HNE, but due to differences in rate, inhibition of PR3 by SerpinB1(S/DAR) is only minimally affected by the presence of HNE even when the latter is in excess. SerpinB1(S/DAR) inhibits soluble PR3 and also membrane-bound PR3 at the surface of activated neutrophils. Moreover, SerpinB1(S/DAR) clears induced PR3 from the surface of activated neutrophils. Overall, these specific inhibitors of PR3 will be valuable for defining biological functions of the protease and may prove useful as therapeutics for PR3-related inflammatory diseases, such as Wegener's granulomatosis.

Proteinase 3 (Pr3), the main target of anti-neutrophil cytoplasmic antibodies, is a neutrophil serine protease that may be constitutively expressed at the surface of quiescent circulating neutrophils. This raises the question of the simultaneous presence in the circulation of constitutive membrane-bound Pr3 (mPr3) and its plasma inhibitor alpha1-protease inhibitor (alpha1-Pi). We have looked at the fate of constitutive mPr3 at the surface of circulating blood neutrophils and of induced mPr3 on triggered neutrophils. We found significant Pr3 activity at the surface of activated neutrophils but not at the surface of quiescent neutrophils whatever the constitutive expression. This suggests that constitutive mPr3 is enzymatically inactive or its active site is not accessible to the substrate. Supporting this conclusion, we have not been able to demonstrate any interaction between constitutive mPr3 and alpha1-Pi, whereas induced mPr3 is cleared from the cell surface when activated cells are incubated with this inhibitor. But, unlike membrane-bound elastase that is also cleared from the surface of activated cells, mPr3 remained bound to the membrane when inhibited by elafin or by a low molecular weight chloromethyl ketone inhibitor, which shows that it binds more tightly to the neutrophil membrane. mPr3 may thus be present at the surface of circulating neutrophils in an environment replete with alpha1-Pi. The permanent presence of inactive Pr3 at the surface of quiescent neutrophils may explain why Pr3 is a major target of anti-neutrophil cytoplasmic antibodies, whose binding activates neutrophils and triggers inflammation, as in Wegener granulomatosis.

The neutrophil serine proteases (NSPs) elastase, proteinase 3 and cathepsin G are multifunctional proteases involved in pathogen destruction and the modulation of inflammatory processes. A fraction of secreted NSPs remains bound to the external plasma membrane, where they remain enzymatically active. This protocol describes the spectrofluorometric measurement of NSP activities on neutrophil surfaces using highly sensitive Abz-peptidyl-EDDnp fluorescence resonance energy transfer (FRET) substrates that fully discriminate between the three human NSPs. We describe FRET substrate synthesis, neutrophil purification and handling, and kinetic experiments on quiescent and activated cells. These are used to measure subnanomolar concentrations of membrane-bound or free NSPs in low-binding microplates and to quantify the activities of individual proteases in biological fluids like expectorations and bronchoalveolar lavages. The whole procedure, including neutrophil purification and kinetic measurements, can be done in 4-5 h and should not be longer because of the lifetime of neutrophils. Using this protocol will help identify the contributions of individual NSPs to the development of inflammatory diseases and may reveal these proteases to be targets for therapeutic inhibitors.

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.